PME-1 as a biomarker to predict and diagnose an increased risk of endometrial cancer and gene silencing of PME-1 to inhibit epithelial to mesenchymal transition

ABSTRACT

Disclosed are methods of attenuating activity of the PME-1 gene. siRNAs or shRNAs are used to target against PME-1, thereby reducing the PME-1 mRNA. It is disclosed that the siRNAs or shRNAs targeted against PME-1 attenuate the epithelial to mesenchymal transition, thereby inhibit endometrial cancer development. A kit containing siRNA or shRNA reagents for attenuating the PME-1 gene expression is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 61/816,472 filed Apr. 26, 2013 andProvisional Application No. 61/905,947 filed Nov. 19, 2013, the contentof which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention generally relates to the discovery that PME-1represents a novel endometrial cancer biomarker (PME-1 is a biomarkerused to predict and diagnose an increased risk of endometrial cancer)and gene silencing of the PME-1 gene to inhibit the epithelial tomesenchymal transition and endometrial cancer development.

BACKGROUND OF THE INVENTION

Endometrial cancer refers to the malignancies of endometrium of theuterus. It is the most common gynecologic cancers in the United States,with over 35,000 women diagnosed each year. It represents the third mostcommon cause of gynecologic cancer death, behind ovarian and cervicalcancer (Siegel, Ca Cancer J Clin. 63:11-30 (2013)). Endometrioidadenocarcinoma often develops in the setting of endometrial hyperplasia,and presents with vaginal bleeding. The most common therapeutic approachof endometrial cancer is a total abdominal hysterectomy with bilateralsalpingo-oophorectomy.

When discovered in its early stages, endometrial cancer is highlycurable (Engelsen, APMIS 117: 693-707 (2009)). There are presentlyseveral methods of clinical evaluation for the presence of endometrialcancer, including Pap smear. Notably, Pap smear is ineffective indetecting endometrial cancer, while it is useful in screening forcervical cancer. Office endometrial biopsy still remains the traditionaldiagnostic method, in which both endometrial and endocervical materialsare often sampled. In the event that endometrial biopsy does not yieldsufficient diagnostic material, a dilation and curettage (D&C) isnecessary for diagnosing the cancer. Hysteroscopy permits the directvisualization of the uterine cavity and can be used to detect thepresence of lesions or tumors. However, such procedure is not practicalbecause it requires strict sterile environments and physician'scompetent skill, as well as the high associated costs.

Transvaginal ultrasound is a non-invasive method that is used toevaluate the endometrial thickness in women with postmenopausal bleedingand is increasingly being used to evaluate for endometrial cancer.However, the method is often not sensitive to detect early endometrialcancer. Studies suggest measurement of serum p53 antibody may be used toidentify high-risk endometrial cancer.

There are currently no other specific biomarkers that exist forendometrial cancer as diagnosis is usually dependent on histology.Several potential biomarkers have been correlated to endometrial cancerin studies, but often these results are inconsistent (Engelsen, APMIS117: 693-707 (2009)).

There is a continuing need in identifying potential biomarker forendometrial cancer in human and means to reduce the epithelial tomesenchymal transition so as to reduce tumor metastasis.

SUMMARY OF THE INVENTION

The present invention provides the use of Protein methyl esterase 1(PME-1) as a biomarker for endometrial cancer, as evidenced by thecorrelation between PME-1 expression levels and endometrial cancer. Thepresent invention further provides methods, such as real-time PCR, forquantifying the expression level of PME-1. The application of thecorrelation between increased levels of PME-1 expression and endometrialcancer is useful in the diagnosis of endometrial cancer. An increasedrisk of endometrial cancer is evidenced when there is an increasedexpression of PME-1 (either protein or mRNA) in a woman with endometrialcancer relative to that of a normal woman (free from endometrialcancer).

In one aspect, the present invention provides characterization the PME-1gene and identification of PME-1 protein that is associated withdevelopment of endometrial cancer. It is discovered that the PME-1expression level in endometrial tissues correlates with the higherstages (i.e., stages II and III) of endometrial cancer in humans.

In one aspect, the present invention provides characterization the PME-1gene and identification of PME-1 protein that is associated withdevelopment of endometrial cancer. It is discovered that the PME-1expression level in endometrial tissues correlates with the highergrades (i.e., stages II and III) of endometrial cancer in humans.

In one aspect, the present invention provides using PME-1 as a biomarkerin endometrial cancer and further provides a novel approach to use RNAinterference (RNAi) (includes siRNA or shRNA) to influence the PME-1expression and thus altering the cancer pathogenesis in human.

In one aspect, the present invention provides using PME-1 proteinexpression level (compared with normal subjects) (i.e., women with nohistory of endometrial cancer) to predict or diagnose endometrial cancerin an individual. An increased risk of endometrial cancer is when thereis an increased PME-1 expression level when comparing a woman at riskrelative to a normal individual.

In one aspect, the present invention provides a method of using RNAi(e.g., siRNA or shRNA) approaches targeting against the PME-1 expressionin human so as to attenuate the epithelial meschenymal transition andthus attenuating endometrial cancer pathogenesis in women.

In one aspect, the present invention provides RNAi as well ascompositions containing RNAi for inhibiting the expression of the PME-1gene in a mammal. The present invention further provides compositionsand methods for treating pathological conditions and diseases mediatedby the expression of the PME-1 gene, such as endometrial cancer. TheRNAi of the present invention comprises an RNA strand (the anti-sensestrand) having a region sufficient to hybridize to mRNA of PME-1(preferably, the 3-UTR of the mRNA) and causes PME-1 mRNA to degrade.Preferably, the RNAi (i.e., siRNA or shRNA) is more than 15 nucleotidesand less than 30 nucleotides in length, generally 20-25 nucleotides inlength, and is substantially complementary to at least part of an mRNAtranscript of the PME-1 gene.

In one aspect, the present invention provides a method of inhibiting thegene expression of PME-1 in a endometrial cell, comprising the steps of:a) providing a RNAi (siRNA or shRNA) targeted against PME-1 mRNA; b)exposing the RNAi to a cell suspected of developing into endometrialtumor, wherein said RNAi inhibits the gene expression of PME-1 in saidendometrial cell.

In one aspect, the present invention provides a method of inhibiting theepithelial to mesenchymal transition (EMT) in a endometrial cellsuspected of developing into a cancerous endometrial cell, comprisingthe steps of: a) providing a RNAi (siRNA or shRNA) targeted againstPME-1 mRNA; b) exposing the RNAi to a cell suspected of developing intoa cancerous endometrial cell, wherein said RNAi inhibits the EMT, asevidenced by a decrease in expression level of E-cadherin, vimentin orfoci formation in said endometrial cell.

In one aspect, the present invention provides a method for inhibitingendometrial cancer progression in a human, comprising the steps ofadministering to a human suspected of suffering from an endometrialcancer an effective amount of a RNAi (siRNA or shRNA) targeted againstPME-1 mRNA, wherein the RNAi inhibits endometrial cancer progression.

In one aspect, the present invention provides a method of detecting anincreased expression level of PME-1 in an endometrial tissue obtainedfrom a woman suspected of suffering from endometrial cancer, comprisingthe steps of: (a) obtaining an endometrial tissue from a woman suspectedof suffering from endometrial cancer; (b) preparing a lysate from saidendometrial tissue; and (c) quantifying an expression level of PME-1protein in said prepared lysate. The increased expression level of PME-1protein relative to that of a normal endometrial tissue is indicative ofan increased risk in endometrial cancer in said woman. Preferably, theexpression level of protein can be quantified using Western blotanalysis or ELISA. Expression mRNA level can be measured using qRT-PCR.Preferably, the total RNA is isolated using guanidinium thiocyanate orphenol-chloroform. The increased PME-1 expression level is associatedwith higher grades of endometrial cancer (i.e., grade II or grade III).

In one aspect, the present invention provides a kit for detectingendometrial cancer in a human, comprising: a) a reagent for quantifyingPME-1 expression level; and b) an instruction for use of said reagent inquantifying PME-1 protein expression level. An increased PME-1 proteinexpression level is indicative of an increased risk in endometrialcancer.

In one aspect, the present invention provides a method of inhibitingepithelial to mesenchymal transition of an endometrial cell, comprisingthe steps of i) providing a RNAi targeted against PME-1 gene, said RNAihybridizes to a target sequence of PME-1 mRNA; and (ii) exposing saidRNAi to an endometrial cell. The RNAi inhibit said epithelial tomesenchymal transition as evidenced by at least one of the featureselected from the group consisting of reduced E-cadherin expression,reduced vimentin expression and reduced foci formation. Preferably, theRNAi is siRNA or shRNA. Preferably, the RNAi is SEQ ID NO: 2, 3, 4, 5,or 6.

In one aspect, the present invention provides a method for inhibitingepithelial to mesenchymal transition in endometrial cells of a womansuspected of suffering from endometrial cancer, comprising the step ofadministering to said woman an effective amount of a RNAi targetedagainst PME-1 gene, whereby said RNAi inhibits PME-1 gene expression soas to inhibit epithelial to mesenchymal transition in endometrial cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Western blot depicting the basal expression of PME-1protein compared to GAPDH (loading control) for (i) the immortalizedendocervical cell line (End1), (ii) the non-aggressive endometrialcancer cell line (KLE), and (iii) the highly aggressive endometrialcancer cell lines, (ECC-1 and RL95-2).

FIG. 2 shows another Western blot depicting the basal expression ofPME-1 protein compared to GAPDH (loading control) for (i) theimmortalized endocervical cell line (End1) and (ii) the endometrialcancer cell lines (RL95-2, ECC-1, Ishikawa). ECC-1 and Ishikawa celllines are closely related but ECC-1 is more aggressive than Ishikawa.

FIG. 3 shows a Western blot depicting a representative panel ofendometrial cancer patient samples examining the levels of PME-1 proteinin tumor (T) and normal adjacent tissue (NAT, N) by Stage. The tumor andnormal adjacent tissue samples were harvested from the same patient.Patient information is available in Table 1.

FIG. 4 shows a Western blot depicting a representative panel ofendometrial cancer patient samples examining the levels of PME-1 proteinin tumor (T) and normal adjacent tissue (NAT, N) by grade. The tumor andnormal adjacent tissue samples were harvested from the same patient.Patient information is available in Table 1.

FIG. 5 depicts immune-histochemistry of patient tissue samplesrepresenting the morphology of normal endometrial tissues (i, v) andendometrial cancers at different stages (i.e., Stages I-III, (ii-iv))and the different expression patterns of PME-1 as the endometrialcancer's stage progresses (i.e., vi-viii).

FIG. 6 depicts immune-histochemistry of patient tissue samplesrepresenting the morphological changes in endometrial tissues asendometrial cancer grade increases (top panels). The hematoxylin andeosin staining (H&E staining) in combination with anti-PME-1immunohistochemistry (IHC, bottom panels) depict the grading of samplesbased on PME-1 levels. The PME-1 grading system is as follows: 0indicates negative cytoplasmic PME-1 staining or faint staining observedin <50% of the cells; 1+ indicates weakly positive cytoplasmic PME-1staining in >50% of tumor cells or moderate/strong staining in <50% oftumor cells; 2+ indicates strongly positive staining withmoderate/strong cytoplasmic staining observed in >50% of the tumorcells. Representative images for PME-1 grading are shown. The tablesummarizes the data obtained from the tissue samples examined by IHC.

FIG. 7A depicts a quantitative PCR of the levels of PPME1 expression intissue samples. PME-1 gene (i.e., PPME1) is the gene that codes forPME-1 protein. Each sample was normalized to the expression of 18S. Allsamples were then normalized to RL95-2 for comparison.

FIG. 7B depicts a ROC analysis of the data set in FIG. 4A, demonstratingPPME1 levels may be predictive of endometrial cancer. Note that the areaunder the curve is >0.9, with a likelihood ratio of 21, the sensitivitywas calculated to be 80.77%, and the specificity 96.15%.

FIG. 8 shows a Western blot of RL95-2 cells depicting the expressingempty vector (Empty), the over-expressing PME-1 (+PME-1) and theexpressing shRNA against PME-1 (shRNA #1, #2 SEQ ID NO: 1 and 2,respectively) demonstrating the proper levels of PME-1 protein. Notethat shRNA #2 (SEQ ID NO: 2) was used for all subsequent experiments.shRNA sequences are presented in Table 2.

FIG. 9A depicts RL95-2 cells (a human endometrial cell line) stablytransfected with a control shRNA (non-targeting) and a shRNA targetedagainst PPME1 (the gene that codes for PME-1). The RL95-2 cells(post-transfection) were collected to analyze changes of PP2Aphosphatase activity. Depletion of PPME1 induced by shRNA targetedagainst PPME1 leads to a ˜30-fold increase in PP2A phosphatase activity,demonstrating that PME-1 protein is an inhibitor of PP2A activity.

FIG. 9B depicts RL95-2 cells that were treated with empty vector(control) or over-expressed with PME-1 protein (+PME-1). The RL95-2cells were collected to analyze changes of PP2A phosphatase activity.Increased PPME1 leads to a 90% decrease in PP2A activity, demonstratingthat PME-1 is an inhibitor of PP2A activity.

FIG. 10A depicts the foci growth of RL95-2 cells expressing empty vector(Empty), over-expressing PME-1 (+PME-1) and expressing shRNA againstPME-1 (−PME-1) (SEQ ID NO: 2) **p<0.01,***p<0.001.

FIG. 10B depicts RL95-2 cells treated with control siRNA (non-targeting)and siRNA against the 3′-UTR of the PME-1 gene (3′-UTR) (SEQ ID NO: 5)to deplete cells of endogenous PME-1 levels. The cells were transfectedwith empty vector (Empty) and a vector over-expressing the inactive formof PME-1 (PME-1 S156A). Cell proliferation of RL95-2 was then assayedvia foci growth. siRNA sequences are presented in Table 2. *p<0.05,**p<0.01

FIG. 11 depicts RL95-2 cells stably expressing control vector (Control),over-expressing PME-1 (+PME-1), or expressing shRNA against PME-1(−PME-1, SEQ ID NO: 2) that were subjected to a Bromodeoxyuridine (BrdU)incorporation assay, which monitors the rate of cell proliferation. BrdUis incorporated into newly synthesized DNA and can be passed intodaughter cells and is a direct measure of cell proliferation. Error barsrepresenting SEM and the significance were calculated by the standardstudent's t test; where *p<0.05, **p<0.01, ***p<0.001.

FIG. 12 depicts stained DAPI (blue) RL95-2 cells expressing empty vector(Empty), over-expressing PME-1 (+PME-1) and expressing shRNA againstPME-1 (−PME-1, SEQ ID NO: 2). The cells were also analyzed byimmunofluorescence, in which an antibody against phosphorylated histoneH3 (red) was used to identify mitotic or proliferating cells.

FIG. 13A depicts RL95-2 cells treated with control siRNA and siRNAtargeted against PME-1 (SEQ ID NO: 4). These cells were subjected to aTUNEL assay to detect apoptosis or fragmented DNA. A positive controlwas included, in which cells were treated with DNase I.

FIG. 13B depicts RL95-2 cells expressing empty vector (control),over-expression of PME-1 (+PME-1) and the expression of shRNA targetedagainst PME-1 (−PME-1). A Western blot analysis was performed probingfor DcR2, a marker of senescent cells, compared to the loading controlGAPDH.

FIG. 14A depicts human endometrial tissue sections immunostained with ananti-PME-1 antibody (green), anti-P-cadherin antibody (red) and DAPInuclear stain (blue). Merged images of red and green staining depictcells that express both markers, indicated by yellow color and arrows.

FIG. 14B depicts human endometrial tissue sections immunostained with ananti-PME-1 antibody (green), anti-E-cadherin antibody (red) and DAPInuclear stain (blue). Merged images of red and green staining depictcells that express both markers, indicated by yellow color and arrows.

Representative images are shown in FIG. 14 from patients diagnosed withgrade 1 endometrioid adenocarcinoma (06313) or grade 3 endometrioidadenocarcinoma (06294). P-cadherin is a marker for aggressiveendometrial cancer (FIG. 14A) and E-cadherin is a marker for cellsretaining epithelial characteristics (FIG. 14B).

FIG. 15 depicts RL95-2 cells expressing empty vector (control),over-expressing PME-1 (+PME-1), and expressing shRNA against PME-1(−PME-1) that were grown on Matrigel to determine the effects of PME-1on invasive growth cancer phenotypes. The colonies of cells werecounted, the means are shown, with error bars representing SEM and thesignificance was calculated by the standard student's t test; where*p<0.05, **p<0.01, ***p<0.001.

FIG. 16 depicts the growth of RL95-2 cells in Matrigel for 10 days inthe presence and absence of TGF-β (which stimulates EMT). The sameamount of RL95-2 cells were also plated and the expression of the emptyvector (control), the over-expression of PME-1 (+PME-1), and theexpression of shRNA against PME-1 (−PME-1) are shown.

FIG. 17 depicts the immunofluorescence of RL95-2 cells expressing emptyvector (control) and over-expressing PME-1 (+PME-1). FIG. 13A depictscells stained with DAPI (blue, nuclear marker) and treated with α-PME-1antibodies to confirm over-expression of PME-1 (compare A_(ii) toA_(i)). FIG. 13B depicts RL95-2 cells treated with α-phospho-ERK(B_(i, ii)) and α-ERK (B_(iii-iv)) to determine if PME-1 over-expressioncorrelates with increased ERK phosphorylation (compare B_(ii) to B_(i)).

FIG. 18A depicts a Western blot of RL95-2 cells expressing empty vector(control), over-expressing PME-1 (+PME-1) and expressing shRNA to PME-1(−PME-1). FIG. 14A depicts RL95-2 cells treated with an up-streaminhibitor of ERK phosphorylation (UO126, +) and a vehicle (−). Lysateswere probed for PME-1, phospho-ERK, and total ERK levels (GAPDH servedas a loading control).

FIG. 18B depicts RL95-2 cells treated with an upstream inhibitor of AKTphosphorylation (LY294002, +) and vehicle (−). Lysates were probed forPME-1 and phosphorylation −AKT at threonine 308 and serine 473 (GAPDHserved as a loading control).

FIG. 19A depicts RL95-2 lysates that were analyzed for total β-cateninlevels when PME-1 was over-expressed (+PME-1) and when PME-1 wasdepleted (−PME-1) in the absence of the proteasome inhibitor, MG-132.

FIG. 19B depicts RL95-2 lysates that were analyzed for β-catenin atserine 45, 33, 37, and threonine 41 when PME-1 was over-expressed(+PME-1) and when PME-1 was depleted (−PME-1) in the presence of theproteasome inhibitor, MG-132.

FIG. 20 depicts RL95-2 cells expressing empty vector (Control) andover-expressing PME-1 (+PME-1). The RL95-2 cells were treated withvehicle or TGF-β for 24 hours and were then analyzed by quantitativePCR. Expression of the epithelial marker, E-cadherin (FIG. 20A), themesenchymal markers, vimentin (FIG. 20B) and Noggin (FIG. 20C), andPPME1 (FIG. 20D) were analyzed. All data was normalized to GAPDHexpression (FIG. 20E).

FIGS. 21A-F depict RL95-2 cells stably expressing control vectors (Emptyor shSCR), over-expressing PME-1 (+PME-1), or expressing shRNA againstPME-1 (shPPME1) (SEQ ID NO: 2). qRT-PCR analysis was completednormalizing the expression of target genes to that of the 18Shouse-keeping gene.

FIG. 21A shows that PME-1 was over-expressed ˜30-fold in +PME-1 cellscompared to empty vector.

FIG. 21B shows that PME-1 was knocked down >50% in cells expressingshPPME1 (SEQ ID NO: 2).

FIG. 21C demonstrates that over-expression leads to a significantdecrease in expression of the SFRP1 gene.

FIG. 21D shows that depletion of PME-1 with shRNA (SEQ ID NO: 2) leadsto a significant increase in SFRP1 expression. SFRP1 is an inhibitor ofWnt signaling.

FIG. 21E demonstrates that over-expression of PME-1 leads to significantincreases in expression of WNT3 and WNT3A genes.

FIG. 21F shows that depletion of PME-1 leads to significantly decreasedWNT3 and WNT3A expression. WNT3 and WNT3A are activators of Wntsignaling.

FIG. 22 depicts ECC-1 cells that were stably transfected with a emptyvector (FLAG), FLAG-tagged PME-1 (over-expression of wild-type PME-1),or FLAG-tagged PME-1 S156A (over-expression of mutated serine (S) 156 toalanine (A), such PME-1 mutant is catalytically inactive). Cells(post-transfection) were collected and cellular protein was extracted.Input depicts the relative expression of each protein, whereas theelution depicts proteins that were immunoprecipitated with FLAG resin.

FIG. 22A shows that FLAG-PME-1 and FLAG-PME-1 S156A wereimmunoprecipitated from the protein lysate. Endogenous Ppp2ca, the alphaisoform of the catalytic subunit of PP2A, binds FLAG-PME-1 (weak) andthe S156A mutant (strong).

FIG. 22B shows that FLAG-PME-1 and FLAG-PME-1 S156A wereimmunoprecipitated from the EEC-1 protein lysate. Endogenous Ppp4c, thecatalytic subunit of PP4, binds PME-1 and PME-1 S156A. Endogenous Ppp6c,the catalytic subunit of PP6, does not bind to PME-1 or PME-1 S156A.

FIGS. 23A-B depict HEK293T cells that were co-transfected with V5 emptyvector, V5-PME-1, or V5-PME-1 S156A in combination with FLAG emptyvector, FLAG-Ppp2ca, or FLAG-Ppp4c.

FIG. 23A depicts the input demonstrating that wild type PME-1 and PME-1S156A were equally expressed and that Ppp2ca and Ppp4c were expressed ata similar level.

FIG. 23B depicts the FLAG elution where Ppp2ca and Ppp4c wereimmunoprecipitated and similar levels of protein were collected. Westernanalysis with the α-V5 antibody shows that PME-1 S156A binds morestrongly to Ppp2ca over Ppp4c, suggesting that PME-1 has a higheraffinity for PP2A than PP4.

FIG. 24 depicts results from a study in which stable clones of ECC-1cells expressing non-targeting scrambled shRNA (shSCR, black bars) orshPPME-1 (white bars) (SEQ ID NO: 2) and were then transfected with FLAGempty vector, FLAG-PPP2CA, FLAG-PPP4C, or FLAG-PPP6C. Cells (3,000) wereplated and were allowed to grow for 10 days. Colonies were then stainedwith crystal violet and counted and the control, cells expressing shSCRand transfected with empty FLAG vector, was normalized to 100% fociformation. PME-1 inhibition via shRNA led to a significant decrease infoci formed regardless of whether PP2A, PP4, or PP6 activity wasincreased. Averages of 6 experiments are plotted and statisticalsignificance was calculated with the Student's T test, where *p<0.05,**p<0.01, ***p<0.001.

FIG. 25 depicts results from a thermal shift assay in which recombinantPME-1 was incubated with either vehicle (circles) or a covalentinhibitor of PME-1, ABL-127 (squares ((3R)-Dimethyl3-cyclopentyl-4-oxo-3-phenyl-1,2-diazetidine-1,2,-dicarboxylate,C₁₇H₂₀N₂O₅, Sigma cat# SML-0294, Bachovchin, PNAS 108: 6811-6816(2011)). Dissociation curves demonstrate that the binding of ABL-127 toPME-1 increases the melting temperature (Tm) and the thermal stabilityof PME-1.

FIG. 26 depicts results from a foci formation assay using DMSO asvehicle and treating Ishikawa cells with the covalent inhibitors ABL-127(50 nM) or AMZ-30 (25 μM)((E)-2-(4-Fluorophenylsulfonyl)-3-(1-(3-nitrophenylsulfonyl)-1H-pyrrol-2-yl)-acrylonitrile),C₁₉H₁₃FN₃O₆S₂, EMD Millipore cat#539695 and Bachovchin, J. Med. Chem.(2011)) every 2-3 days for a total of 10 days. shSCR and shPPME1 treatedcells are used for comparison and were normalized to untreated treatedcells (not shown) set to 100%.

FIG. 27 depicts results from a trans-well migration assay using DMSO asvehicle and treating Ishikawa cells with 50 nM ABL-127 or 20 μM AMZ-30for 24 hr after synchronizing cells in 0% FBS for 24 hr. Cells migratedthrough collagen towards the bottom well containing 30% FBS.

FIGS. 28A-D depicts results from a mice xenograft study. 1×10⁶ ECC-1cells expressing empty vector (control) or over-expressing PME-1(+PME-1) were injected subcutaneously into the flank ofimmune-compromised mice (n=7 per group).

FIG. 28A depicts a Western blot of ECC-1 cells prior to injection,confirming levels of PME-1 over-expression when compared to GAPDH.

FIG. 28B depicts the tumor volume developed in the immune-compromisedmice over 7 weeks. The tumors were measured (length, y, and width, x)weekly for 7 weeks and the volume of each tumor was calculated using theequation V=½(yx²). The means were plotted with error bars representingSEM and significance was calculated using two-way ANOVA where **p<0.01,***p<0.001.

FIG. 28C depicts representative tumors resected at week 8 (the whitebars represent 0.5 cm).

FIG. 28D depicts a graphical representation of the tumors' weights; theline represents the mean tumor weight per group at week 8. Note that twomice from the control group did not form tumors.

FIG. 29 depicts the results from a mice xenograft study. 5×10⁶ ofIshikawa cells with Matrigel were injected into the flank of female SCIDmice and the tumors were allowed to grow to ˜400 mm³. Tumors were theninjected intratumorally every 3-4 days with 5×10⁷ pfu of adenovirus,delivering either scrambled shRNA, closed circles, or PME-1 shRNA (SEQID NO: 3), open circles. Depletion of PME-1 significantly decreasedtumor volume by day 13 after treatment was initiated. Data is shown asfold-change in tumor volume from the start of treatment. The means wereplotted with error bars representing SEM and significance was calculatedusing two-way ANOVA where **p<0.01, ***p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be better understood from the followingdescription of preferred embodiments, taken in conjunction with theaccompanying drawings. It should be apparent to those skilled in the artthat the described embodiments of the present invention provided hereinare merely exemplary and illustrative and not limiting.

DEFINITIONS

Various terms used throughout this specification shall have thedefinitions set out herein.

As used herein, the term “A,” “T,” “C”, “G” and “U” refer to adenine,thymine, cytosine, guanine, uracil as a nucleotide base, respectively.

As used herein the term “RNAi” refers to RNA interference. For purposesof this application, RNAi encompasses siRNA and shRNA.

As used herein, the term “siRNA” refers to a small interfering RNA. RNAinterference refers to the process of sequence-specificpost-transcriptional gene silencing in a cell or an animal mediated byshort interfering RNA.

As used herein, the term “shRNA” refers to small hairpin RNA comprisedof complimentary sequences that produce a hairpin shape. shRNA isprocessed within the cell to produce a target sequence that specificallypromotes degradation of PPME1 mRNA via sequence-specific complementarybase pairings.

As used herein, the term “shSCR” refers to small hairpin RNA comprisedof “scrambled” or “non-targeting” sequences that are designed in such away that their presence does not affect the mRNA stability of any humangene and therefore no gene expression is affected. shSCR is used as acontrol.

As used herein, the term “siRNA or shRNA targeted against PME-1” refersto siRNA or shRNA specifically promote degradation of PPME1 mRNA viasequence-specific complementary base pairings.

As used herein, the term “target sequence” refers to a contiguousportion of the nucleotide sequence of an mRNA molecule (e.g., 3-UTR ofan mRNA molecule) of a particular gene (i.e., PPME1 gene). The targetsequence of a siRNA of the present invention refers to an mRNA sequenceof that gene that is targeted by the siRNA by virtue of itscomplementarity of the anti-sense strand of the siRNA to such sequenceand to which the anti-sense strand hybridizes when brought into contactwith the mRNA. The mRNA sequence may include the number of nucleotidesin the anti-sense strand as well as the number of nucleotides in asingle-stranded overhang of the sense strand, if any.

As used herein, the term “complementary” refers to the ability of afirst polynucleotide to hybridize with a second polynucleotide.

As used herein, the term “introducing into a cell”, when referring to adsRNA, means facilitating uptake or absorption into the cell, as isunderstood by those skilled in the art. Absorption or uptake of dsRNAcan occur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. The meaning of this term is not limited tocells in vitro; a dsRNA may also be “introduced into a cell”, whereinthe cell is part of a living organism. In such instance, introductioninto the cell will include the delivery to the organism. For example,for in vivo delivery, dsRNA can be injected into a tissue site oradministered systemically. In vitro introduction into a cell includesmethods known in the art such as electroporation and lipofection.

As used herein, the terms “silencing” and “inhibiting the expressionof”, in as far as they refer to the PPME1 gene refers to at leastpartial suppression of the expression of the PPME1 gene, as manifestedby a reduction of the amount of mRNA transcribed from the PPME1 gene.Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to PPME1 genetranscription, e.g. the amount of protein encoded by the PPME1 genewhich is secreted by a cell or found in solution after lysis of suchcells.

As used herein, the term “PPME1” refers to the PME-1 gene (i.e., theprotein phosphatase methylesterase 1 gene), the nucleotide sequence ofwhich is listed under Genbank accession numbers NM_016147.1, thedisclosure of which is incorporated herein by reference.

As used herein, the term “PME-1” refers to the protein that istranslated from the PPME1 gene and is a demethylase (also known as amethylesterase) that removes a methyl group post-translationalmodification from another protein.

As used herein, the term “demethylase” or “methylesterase” refers to aprotein capable of removing a methyl group (or demethylating) anotherprotein.

As used herein, the term “protein phosphatase 2A (PP2A)” refers to aprotein complex that is capable of removing a phosphate group fromanother protein. PP2A is comprised of two to three different proteins.The core complex is comprised of a catalytic subunit and a scaffoldingsubunit. The addition of a regulatory subunit targets the complex to aspecific substrate.

As used herein, the term “metastasis” refers to cancer cells migratingaway from a solid tumor and the establishment of a new tumor in asecondary site.

As used herein, the term “RL95-2” and “ECC-1” and “Ishikawa” refer tohuman endometrial cancer cell lines that are highly aggressive andcapable of metastasis.

As used herein, the term “KLE” refers to a human endometrial cancer cellline that is not highly aggressive.

As used herein, the term “End1” refers to a human immortalizedendocervical cell line.

As used herein, the term “WNT3” and “WNT3A” refer to related members ofthe Wingless Type MMTV Integration site family (WNT) members of proteinsthat bind to and activate frizzled receptors that promotes increasedgene expression of genes related to cell proliferation and epithelial tomesenchymal transition and metastasis. Wnt proteins activate thispathway and are commonly expressed to higher levels in cancers.

As used herein, the term “SFRP1” refers to secreted frizzled-likeprotein 1, which is binds to Wnt and Frizzled proteins and inhibits theWnt signaling pathway.

As used herein, the term “foci” and “foci formation” refer to an assayin which a known number of cells are plated in a tissue culture dish andare allowed to grow for a defined number of days and form collections ofcells—or foci—that can be stained and counted to quantify cell growth.

As used herein, the term “BrdU assay” refers to an assay in which cellsare incubated with bromodeoxyuridine (BrdU), which is incorporated intonewly synthesized DNA. BrdU can be detected with an antibody to directlymeasure the rate of cellular proliferation.

As used herein, the term “endometrial cancer” refers to the cancer thatstarts at the endometrium, the tissue lining of the uterus. Endometrialcancer is also referred to as uterine cancer.

As used herein, the term “stage I” refers to endometrial cancer that isconfined to the uterus.

As used herein, the term “stage II” refers to endometrial cancer thathas spread from the uterus to the cervix (the lower part of the uterus)but has not spread outside the uterus.

As used herein, the term “stage III” refers to endometrial cancer thathas spread to one or more of the following: the outermost layer of theuterus (uterine serosa), the tissue just beyond the uterus (i.e.adnexa—tissues on either side of the uterus) or the ovary.

As used herein, the term “FIGO grading” refers to the grading system forcarcinoma of the endometrium developed by the International Federationof Gynecology and Obstetrics. The FIGO system is most commonly used andis the required grading system for carcinoma of the endometrium underthe College of American Pathologists' protocol. FIGO grading excludesserous or clear cells, which are considered high grade (grade 3).Alternative grading divides endometrioid tumors into low grade or highgrade based on solid growth (50% or less vs. 50%+), pattern of invasion(infiltrative vs. expansive) and presence of tumor cell necrosis (yesvs. no). A patient will be diagnosed with high grade if the tumors cellshave 2 of these features.

As used herein, the term “grade 1” or “FIGO Grade 1” refers toendometrial cells that resemble microglandular hyperplasia and arecomposed primarily of well formed glands, having <5% nonsquamous solidcomponent.

As used herein, the term “grade 2” or “FIGO Grade 2” refers toendometrial cells that are composed of between 6% and 50% nonsquamoussolid components.

As used herein, the term “grade 3” or “FIGO Grade 3” refers toendometrial cells that have more than 50% nonsquamous solid component,lack well formed glands, which differentiates it from serous endometrialcarcinoma. As used herein, the term “cell proliferation” or “cellgrowth” refers to the rate at which cells divide and complete the cellcycle.

As used herein, the term “epithelial to mesenchymal transition” or “EMT”refers to cells that are capable of migration due to loss ofcell-to-cell contact, changes in cell morphology, loss of epithelialmarkers, such as E-cadherin, and acquisition of mesenchymal markers,such as vimentin.

As used herein, the term “E-cadherin” refers to a protein that is amember of the cadherin superfamily. E-cadherin is a calcium-dependentadhesion glycoprotein and plays a major role in maintaining cell-to-cellcontacts. Loss of E-cadherin is a hallmark of EMT.

As used herein, the term “P-cadherin” refers to a protein that is amember of the cadherin superfamily and is a transmembrane glycoprotein.Increases in P-cadherin expression has been correlated with increasedaggressivity of endometrial cancers.

As used herein, the term “vimentin” refers to a protein that is a memberof the intermediate filament family and is part of the cytoskeleton.Vimentin plays a role in maintenance of cell shape and organizes otherproteins involved in cell attachment, migration, and cell signaling.Increased vimentin is a hallmark of EMT.

As used herein, the term “Noggin” is a secreted protein that binds andinactivates members of the TGF-β family of signaling proteins. Increasednoggin is a marker for EMT.

As used herein, the term “TGF-β” or “transforming growth factor β”protein is a cytokine that regulates cell proliferation,differentiation, adhesion, and migration and is commonly increased incancer cells. Addition of TGF-β to cells in culture can promote EMT.

As used herein, the term “β-catenin” refers to a protein that is part ofa complex of proteins that are important for maintaining cell-to-cellcontacts and may transmit contact inhibition signals to stop cellgrowth.

As used herein, the term “epithelial” refers to ordered anddifferentiated cells that line the inner and outer surfaces of the body.

As used herein, the term “mesenchymal” refers to cells that are lessdifferentiated and have the ability to differentiate into differenttypes of cells or migrate.

As used herein, the term “extracellular signal-regulated kinase” or“ERK” refers to a protein that is a mitogen-activated protein kinase andsignals through the phosphorylation and activation of other proteins,such as transcription factors, to promote cell proliferation. ERKactivity is often increased in cancer.

As used herein, the term “Akt” is a protein kinase that is part of acellular signaling cascade that promotes cell proliferation.Phosphorylated Akt is active and promotes cell proliferation. Aktactivity is often increased in cancer.

As used herein, the term “apoptosis” is a natural process of cellularsuicide that is marked by the fragmentation of DNA and can occur due toaccumulation of DNA damage.

As used herein, the term “senescence” refers to the process by whichcells exit the cell cycle and cease to proliferate.

As used herein, the term “cell cycle” refers to the process by which acell divides to produce two identical cells. Cells undergo DNAreplication, two periods of growth, and enter mitosis.

As used herein, the term “mitosis” refers to the cellular process inwhich a cell divides to produce two, nearly identical cells.

As used herein, the terms “attenuate”, “treat” and “treatment” refer torelief from or alleviation of pathological processes mediated by PME-1expression. In other words, relief from or alleviate at least onesymptom associated with such condition, or to slow or reverse theprogression of such condition.

The term “inhibit gene expression” refers to the use of siRNA or shRNAmolecule to down regulate the expression of target gene PME-1 mRNA,which thereby leads to reduced expression of the PME-1 gene.

As used herein, the term “quantitative PCR” refers to the quantitativepolymerase chain reaction. Quantitative PCR is a means for quantifyingthe amount of template DNA present in the original mixture, usuallyachieved by the addition of a known amount of a target sequence that isamplified by the same primer set but can be differentiated, usually bysize, at the end of the reaction.

As used herein, the term “real-time PCR” refers to the real-timepolymerase chain reaction. Real-time PCR is a method for the detectionand quantification of an amplified PCR product based on a fluorescentreporter dye; the fluorescent signal increases in direct proportion tothe amount of PCR product produced and is monitored at each cycle, ‘inreal time’, such that the time point at which the first significantincrease in the amount of PCR product correlates with the initial amountof target template.

As used herein, the term “quantitative reverse transcription PCR” (i.e.,“qRT-PCR”) refers to a quantitative polymerase chain reaction (qPCR)used to detect mRNA expression levels. The qRT-PCR contains a first stepwherein the mRNA molecules are converted to complementary DNA molecules(cDNAs) by reverse transcription enzyme in a “reverse transcription”reaction (RT). The qRT-PCR contains a second step wherein the expressionlevels of mRNA are quantified.

As used herein, the term “Ct score” or “Cycle threshold” refers to theamount of PCR cycles required for the accumulation of a fluorescentsignal to reach the threshold level or the amount of PCR cycles requiredto surpass background levels

As used herein, the term “delta Ct” refers to the difference in Ctvalues when the Ct value for the normalizer gene (one that should notchange across experimental conditions, i.e. 18S) is subtracted from Ctvalue for the gene of interest.

As used herein, the term “pharmaceutical composition” comprises apharmacologically effective amount of a RNAi and a pharmaceuticallyacceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier for administration of a therapeutic agent.

As used herein, the term “therapeutically effective amount” refers to anamount that provides a therapeutic benefit in the treatment, prevention,or management of pathological processes.

The present invention provides a novel biomarker for detection ofendometrial cancer. Specifically, the present inventors discovered theexpression level of PME-1 correlates with the endometrial cancerdevelopment. More specifically, the increased expression level of PME-1is associated with higher grades of endometrial cancer (i.e., grade IIand grade III). The present application discloses the use of PME-1 as abiomarker to detect or diagnose endometrial cancer in women.

Using cell cultures of endometrial cells, the present inventors foundthat the expression levels of PME-1 (including both protein and mRNA)are elevated in these cells when compared to their counterparts. Thepresent inventors extended the in vitro finding to clinical application.Using clinical samples obtained from women who suffered from variousstages/grades of endometrial cancer, the present inventors demonstratedthat PME-1 expression levels (protein or mRNA) increase in endometrialtissues derived from patients who suffer from endometrial cancer ascompared to endometrial tissues derived from control patients who do nothave endometrial cancer. The expression levels of PME-1 (either proteinor mRNA) significantly increase throughout higher cancer stages (i.e.,stage II, and stage III of endometrial cancer).

The present invention provides a diagnostic assay having a highsensitivity and specificity for detecting endometrial cancer occurrencein a woman suspected of having endometrial cancer. ROC analysisdemonstrates that the sensitivity of the present assay is 80.77% and thespecificity is 96.15%. PPME1 mRNA expression with a cut-off of 8.14(determined using Prism software) demonstrates a 95.5% positivepredictive value (PPV) and an 83.3% negative predictive value (NPV).

There has been a long-felt need for identifying a novel biomarker thatwould permit early detection and diagnosis of endometrial cancerdiseases. Little is known about biomarkers that may serve for thedetection of endometrial cancer.

The present inventors cured the prior art deficiency and fulfilled thelong-felt need in this medical area. The present inventors surprisinglydiscovered PME-1 as a biomarker for detection of endometrial diseases inhumans. Protein methylesterase-1 (PME-1) is a specific methylesteraseand removes the methyl group from the C-terminal leucine residue on theC subunit of the PP2A that has tumor suppressor activity. The methylpost-translational modification is believed to increase the tumorsuppressor activity of PP2A, a molecule that is involved in numerouscellular processes, including signal transduction pathways that regulatemitogenic and survival signals. In yeast, disruption ofmethyltransferase for methylating PP2A leads to severe growth defects.In mice, PME-1 targeted gene disruption causes perinatal lethality and anear complete loss of demethylated PP2A in most tissues, suggestingPME-1 may play a role in cellular signaling. Because certain forms ofPP2A are known to stabilize p53 activity or decrease the activity ofpro-cell growth signaling pathways, it is possible that PME-1 maycontribute to endometrial cancer initiation and progression. The exactrole PME-1 in cancer progression remains unclear, let alone endometrialcancer.

To the best of the present inventors' knowledge, PME-1 expression hasbeen reported to significantly correlate with disease progression inhuman astrocytic gliomas. The present invention represents the firstreport linking PME-1 expression with endometrial cancer. The presentinvention provides a novel finding that PME-1 is a good molecularbiomarker for detection of endometrial cancer. The present finding isunexpected because the present assay employing PME-1 expression providesa high sensitivity and specificity.

Collection and Preparation Biological Samples

Biological samples of the endometrium in humans (including cells ortissues) can be conveniently collected by methods known in the art.Usually, endometrial tissues (or cells thereof) can be harvested bytrained medical staffs or physicians, under sterile environment.Endometrial tissues or cells may be taken, for example, by scrapes,smears, or swabs. Swabs may include but are not limited to, spatulas,brushes, or brooms. Other means include punch biopsy, endocervicalcurettage, conization, resection of tumor samples, tissue samplesprepared by endoscopic means, needle-biopsies of organs, and the like.After harvested from patients, biological samples may be immediatelyfrozen (under liquid nitrogen) or put into a storage, or transportationsolution to preserve sample integrity. Such solutions are known in theart and commercially available, for example, UTM-RT transport medium(Copan Diagnostic, Inc, Corona, Calif.), Multitrans Culture Collectionand Transport System (Starplex Scientific, Ontario, Conn.), ThinPrep®Paptest Preservcyt® Solution (Cytyc Corp., Boxborough, Mass.) and thelike.

Sample Preparation: Protein Extraction

After collection, tissue samples are prepared prior to detection ofbiomarkers. Sample preparation includes isolation of protein or nucleicacids (e.g., mRNA). These isolation procedures involve separation ofcellular protein or nucleic acids from insoluble components (e.g.,cytoskeleton) and cellular membranes.

In one embodiment, endometrial tissues or cells are treated with a lysisbuffer solution prior to isolation of protein or nucleic acids. A lysisbuffer solution is designed to lyse tissues, cells, lipids and otherbiomolecules potentially present in the raw tissue samples. Generally, alysis buffer of the present invention may contain a chemical agent thatincludes one or more of the following ingredients: (i) chaotropic agents(e.g., urea, guanidine thiocyanide, or formamide); (ii) anionicdetergents (e.g., SDS, N-lauryl sarcosine, sodium deoxycholate, olefinesulphates and sulphonates, alkyl isethionates, or sucrose esters); (iii)cationic detergents (e.g., cetyl trimethylammonium chloride); (iv)non-ionic detergents (e.g., Tween®-20, polyethylene glycol sorbitanmonolaurate, nonidet P-40, Triton® X-100, NP-40, N-octyl-glucoside); (v)amphoteric detergents (e.g., CHAPS,3-dodecyl-dimethylammonio-propane-1-sulfonate, lauryldimethylamineoxide); or (vi) alkali hydroxides (e.g., sodium hydroxide or potassiumhydroxide). Suitable liquids that can solubilize the cellular componentsof biological samples are regarded as a lysis buffer for purposes ofthis application.

In another embodiment, a lysis buffer may contain additional substancesto enhance the properties of the solvent in a lysis buffer (e.g.,prevent degradation of protein or nucleic acid components within the rawbiological samples). Such components may include proteinase inhibitors,RNAse inhibitors, DNAse inhibitors, and the like. Proteinase inhibitorsinclude but not limited to inhibitors against serine proteinases,cysteine proteinases, aspartic proteinases, metallic proteinases, acidicproteinases, alkaline proteinases or neutral proteinases. RNAseinhibitors include common commercially available inhibitors such asSUPERase.In® (Ambion, Inc. Austin, Tx), RNAse Zap® (Ambion, Inc. Austin,Tx), Qiagen RNase inhibitor (Valencia, Calif.), and the like.

Sample Preparation: Nucleic Acid Extraction

Nucleic acids, such as mRNA, can be conveniently extracted frombiological samples obtained from endometrium (i.e., endometrial tissues)using standard extraction methods that are known in the art. Standardextraction methods include the use of a chemical agent such asguanidinium thiocyanate, phenol-chloroform extraction, guanidine-basedextraction, and the like. Commercial nucleic acid extraction kits may beemployed. For example, RNeasy Fibrous Tissue Mini Kit from Qiagen(Valencia, Calif.) and RNAimage Kit from GenHunter Corporation (USA).

Detection of Protein Expression Level

After protein extraction, expression level of PME-1 protein in thebiological samples can be determined using standard assays that areknown in the art. These assays include but not limited to Western blotanalysis, ELISA, radioimmunoassay, fluoroimmunoassay,immunohistochemistry assay, dot-blot assay, and the like. In a preferredembodiment, expression level of biomarkers may be detected by Westernblot analysis. In another preferred embodiment, PME-1 protein expressionmay be determined by Western blot analysis.

Western Blot

After cellular proteins are extracted or isolated from the biologicalsamples (e.g., endometrial tissues), the cellular proteins are separatedusing SDS-PAGE gel electrophoresis. The conditions for SDS-PAGE gelelectrophoresis can be conveniently optimized by one skilled in the art.

Protein biomarkers in the gels can then be transferred onto a surfacesuch as nitrocellulose paper, nylon membrane, PVDF membrane and thelike. The conditions for protein transfer after SDS-PAGE gelelectrophoresis may be optimized by one skilled in the art. Preferably,a PVDF membrane is used.

To detect the biomarker proteins, a first antibody specific for thePME-1 protein is employed. Bound cellular proteins (e.g., 15-100 μg) onthe membrane are incubated with a first antibody in a solution. Anoptimized first antibody concentration (e.g., 0.2-2 μg/mL) may be used.Incubation conditions may be optimized to maximize binding of the firstantibody with the bound biomarker proteins. For example, 1 μg/mL of thefirst antibody is used and incubation time is 1-6 hours. Preferably, theincubation time is 2 hours. The first antibody may either be amonoclonal antibody or polyclonal antibody. Antibodies against thevarious protein biomarkers can be prepared using standard protocols orobtained from commercial sources. Techniques for preparing mousemonoclonal antibodies or goat or rabbit polyclonal antibodies (orfragments thereof) are well known in the art. Optionally, the membraneis incubated with a blocking solution before the incubation with thefirst antibody. The blocking solution may include agents that reducenon-specific binding of antibody. An exemplary blocking solution mayinclude 5% skim milk in PBST (Phosphate Buffer Solution containing 0.1%Tween-20).

After the incubation with the first antibody, the unbound antibody isremoved by washing. An exemplary washing solution includes PBST. Proteinbiomarker-first antibody complex can be detected by incubation with asecond antibody that is specific for the first antibody. The secondantibody may be a monoclonal antibody or a polyclonal antibody (e.g.,mouse, rabbit, or goat). The second antibody may carry a label which maybe a directly detectable label or may be a component of asignal-generating system. Preferably, the second antibody is a goatanti-rabbit antibody or goat anti-mouse antibody that is labeled with aperoxidase. Such labeled antibodies and systems are well known in theart.

Direct detectable label or signal-generating systems are well known inthe field of immunoassay. Labeling of a second antibody with adetectable label or a component of a signal-generating system may becarried out by techniques well known in the art. Examples of directlabels include radioactive labels, enzymes, fluorescent andchemiluminescent substances. Radioactive labels include ¹²⁴I, ¹²⁵I,¹²⁸I, ¹³¹I, and the like. A fluorescent label includes fluorescein,rhodamine, rhodamine derivatives, and the like. Chemiluminescentsubstances include ECL chemiluminescent.

ELISA

In another embodiment, detection and quantification of PME-1 proteinbiomarker is determined by ELISA.

In a typical ELISA, a first antibody is immobilized onto a solidsurface. Immobilization of the first antibody may be performed on anyinert support useful in immunological assays. Examples of inert supportinclude sephadex beads, polyethylene plates, polypropylene plates,polystyrene plates, and the like. In one embodiment, the first antibodyis immobilized by coating the antibody on a microtiter plate. In anotherembodiment, the microtiter plate is a microtest 96-well ELISA plate,such as those sold under the name Nunc Maxisorb or Immulon.

The first antibody is an antibody specific (to bind or to recognize) theprotein biomarkers of interest. The first antibody may either be amonoclonal antibody, polyclonal antibody, or a fragment thereof. Thefirst antibody may be acquired via commercial sources, or prepared bystandard protocols well known in the art. A solid surface includes a96-well plate.

Protein biomarkers present in a biological sample are captured by theimmobilized first antibody. To do so, a protein extract from biologicalsamples is incubated with the immobilized first antibody. Conditions forincubation can be optimized to maximize the formation of proteinbiomarker-first antibody complex. Preferably, an incubation time of 2-8hours and a temperature of 25° C. may be used. Unbound first antibody isremoved by washing.

To detect the formation of protein biomarker-first antibody complex, asecond antibody is used. The second antibody may either be a monoclonalantibody or polyclonal antibody. Preferably, the second antibody is apolyclonal antibody, derived from goat or rabbit. Preparation of thesecond antibody is in accordance with established protocol orcommercially available. Incubation of the second antibody canconveniently be optimized to maximize the binding. Preferably, anincubation time of 2-8 hours and a temperature of 25° C. may be used.Unbound second antibody is easily removed by washing. The secondantibody is either directly labeled or conjugated with asignal-generating system.

The methods of detecting the presence of a directly labeled secondantibody or a second antibody conjugated with a signal-generating systemare well known to those of skill in the art. Suitable direct labelsinclude moieties such as fluorophores, radioactive labels, and the like.Examples of radioactive labels include but not limited to ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I. Examples of fluorophores include but not limited tofluorescein, rhodamine, and the like.

The second antibody may conveniently be conjugated to asignal-generating system such as an enzyme. Exemplary enzymes includehorseradish peroxidase (HRP), alkaline phosphatase, and the like. Theconjugation of an enzyme to the second antibody is a standardmanipulative procedure for one of ordinary skill in immunoassaytechniques. (See, for example, O'Sullivan et al. “Methods for thePreparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166).Detection of the presence of second antibody can be achieved simply byadding a substrate to the enzyme. The methodology of suchenzyme-substrate interaction is well within one skilled in the art'scapability.

Detection of mRNA Expression Level

In one embodiment, the present invention is directed to the discoverythat PME-1 protein biomarker is elevated during the pathogenesis ofendometrial cancer. In another embodiment, endometrial cancer biomarkersincrease their steady-state mRNA expression levels. Detection of mRNAexpression levels for PME-1 gene includes standard mRNA quantitationassays that are well-known in the art. These assays include but notlimited to qRT-PCR, Northern blot analysis, RNase protection assay, andthe like.

In one preferred embodiment, the present invention provides the use ofqRT-PCR to detect the expression level of endometrial cancer biomarkers.qRT-PCR (quantitative reverse transcription-polymerase chain reaction)is a sensitive technique for mRNA detection and quantitation. Comparedto Northern blot analysis and RNase protection assay, qRT-PCR can beused to quantify mRNA levels from much smaller samples.

Real-time polymerase chain reaction, also called quantitative real timepolymerase chain reaction (Q-PCR/qPCR), is used to amplify andsimultaneously quantify a targeted DNA molecule. It enables bothdetection and quantification (as absolute number of copies or relativeamount when normalized to DNA input or additional normalizing genes) ofone or more specific sequences in a DNA sample. Currently at least four(4) different chemistries, TagMan® (Applied Biosystems, Foster City,Calif.), Molecular Beacons, Scorpions® and SYBR® Green (MolecularProbes), are available for real-time PCR.

All of these chemistries allow detection of PCR products via thegeneration of a fluorescent signal. TaqMan probes, Molecular Beacons andScorpions depend on Förster Resonance Energy Transfer (FRET) to generatethe fluorescence signal via the coupling of a fluorogenic dye moleculeand a quencher moiety to the same or different oligonucleotidesubstrates. SYBR Green is a fluorogenic dye that exhibits littlefluorescence when in solution, but emits a strong fluorescent signalupon binding to double-stranded DNA.

Two common methods for detection of products in real-time PCR are: (1)non-specific fluorescent dyes that intercalate with any double-strandedDNA, and (2) sequence-specific DNA probes consisting of oligonucleotidesthat are labeled with a fluorescent reporter which permits detectiononly after hybridization of the probe with its complementary DNA target.

Real-time PCR, when combined with reverse transcription, can be used toquantify messenger RNA (mRNA) in cells or tissues. An initial step inthe reverse transcription PCR amplification is the synthesis of a DNAcopy (i.e., cDNA) of the region to be amplified. Reverse transcriptioncan be carried out as a separate step, or in a homogeneous reversetranscription-polymerase chain reaction (RT-PCR), a modification of thepolymerase chain reaction for amplifying RNA. Reverse transcriptasessuitable for synthesizing a cDNA from the RNA template are well known.

Following the cDNA synthesis, methods suitable for PCR amplification ofribonucleic acids are known in the art (See, Romero and Rotbart inDiagnostic Molecular Biology: Principles and Applications pp. 401-406).PCR reagents and protocols are also available from commercial vendors,such as Roche Molecular Systems. PCR can be performed using an automatedprocess with a PCR machine.

Primer sets used in the present qRT-PCR reactions for various biomarkersmay be prepared or obtained through commercial sources. For purposes ofthis application, the primer sets used in this invention include primersordered from Life Technologies (Assay ID, HS00211693_m1) (Grand Island,N.Y.).

The primers used in the PCR amplification preferably contain at least 15nucleotides to 50 nucleotides in length. More preferably, the primersmay contain 20 nucleotides to 30 nucleotides in length. One skilled inthe art recognizes the optimization of the temperatures of the reactionmixture, number of cycles and number of extensions in the reaction. Theamplified product (i.e., amplicons) can be identified by gelelectrophoresis.

Aided with the help of DNA probe, the real-time PCR provides a quantumleap as a result of real-time detection. In real-time PCR assay, afluorometer and a thermal cycler for the detection of fluorescenceduring the cycling process is used. A computer that communicates withthe real-time machine collects fluorescence data. This data is displayedin a graphical format through software developed for real-time analysis.

In addition to the forward primer and reverse primer (obtained viacommercial sources), a single-stranded hybridization probe is also used.The hybridization probe may be a short oligonucleotide, usually 20-35 bpin length, and is labeled with a fluorescent reporting dye attached toits 5′-end as well as a quencher molecule attached to its 3′-end. When afirst fluorescent moiety is excited with light of a suitable wavelength,the absorbed energy is transferred to a second fluorescent moiety (i.e.,quencher molecule) according to the principles of FRET. Because theprobe is only 20-35 bp long, the reporter dye and quencher are in closeproximity to each other and little fluorescence is detected. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product). At the sametime, Taq DNA polymerase extends from each primer. Because of its 5′ to3′ exonuclease activity, the DNA polymerase cleaves the downstreamhybridization probe during the subsequent elongation phase. As a result,the excited fluorescent moiety and the quencher moiety become spatiallyseparated from one another. As a consequence, upon excitation of thefirst fluorescent moiety in the absence of the quencher, thefluorescence emission from the first fluorescent moiety can be detected.By way of example, a Rotor-Gene System is used and is suitable forperforming the methods described herein. Further information on PCRamplification and detection using a Rotor-Gene can conveniently be foundon Corbett's website.

In another embodiment, suitable hybridization probes such asintercalating dye (e.g., Sybr-Green I) or molecular beacon probes can beused. Intercalating dyes can bind to the minor grove of DNA and yieldfluorescence upon binding to double-strand DNA. Molecular beacon probesare based on a hairpin structure design with a reporter fluorescent dyeon one end and a quencher molecule on the other. The hairpin structurecauses the molecular beacon probe to fold when not hybridized. Thisbrings the reporter and quencher molecules in close proximity with nofluorescence emitted. When the molecular beacon probe hybridizes to thetemplate DNA, the hairpin structure is broken and the reporter dye is nolong quenched and the real-time instrument detects fluorescence.

The range of the primer concentration can optimally be determined. Theoptimization involves performing a dilution series of the primer with afixed amount of DNA template. The primer concentration may be betweenabout 50 nM to 300 nM. An optimal primer concentration for a givenreaction with a DNA template should result in a low Ct-(thresholdconcentration) value with a high increase in fluorescence (5 to 50times) while the reaction without DNA template should give a highCt-value.

The probes and primers of the invention can be synthesized and labeledusing well-known techniques. Oligonucleotides for use as probes andprimers may be chemically synthesized according to the solid phasephosphoramidite triester method first described by Beaucage, S. L. andCaruthers, M. H., 1981, Tetrahedron Letts., 22 (20): 1859-1862 using anautomated synthesizer, as described in Needham-VanDevanter, D. R., etal. 1984, Nucleic Acids Res., 12: 6159-6168. Purification ofoligonucleotides can be performed, e.g., by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson, J. D.and Regnier, F. E., 1983, J. Chrom., 255: 137-149.

Comparison of Expression Levels of PME-1 Endometrial Cancer Biomarker

Expression levels of PME-1 in a biological sample obtained from apatient (suspected with endometrial cancer) may be compared to theexpression levels of PME-1 endometrial cancer biomarker obtained fromnormal endometrial tissues. Normal endometrial tissues includeendometrial tissues obtained from healthy individuals or endometrialtissues obtained from an adjacent area to the cancer regions within theendometrium of the endometrial cancer patients under examination.Comparison may be performed by employing protein concentrations of theendometrial cancer biomarkers, or ΔΔC_(t) values from qRT-PCR of theendometrial cancer biomarker genes.

Specifically, more aggressive endometrial cancer is associated with anincrease in PME-1 expression levels, as compared to normal adjacentendometrial tissue (eg., FIG. 7 where expression of PME-1 is increasedby twenty-fold in endometrial cancer tissue). The correlation betweenPME-1 and endometrial cancer is used to predict or diagnose endometrialcancer in a patient.

In certain embodiments, the step of comparing the expression levels ofendometrial cancer biomarkers (e.g., PME-1) present in a patient sampleto an expression level of the same biomarker known to be present in anormal healthy body sample is embodied as employing a cut-off value orthreshold value for the concentration of that particular biomarker.

Kits

The present invention provides a kit of manufacture, which may be usedto perform detecting either a protein or mRNA for a specific endometrialcancer biomarker. In one embodiment, an article of manufacture (i.e.,kit) according to the present invention includes a set of antibodies(i.e., a first antibody and a second antibody) specific for a PME-1protein biomarker. In another embodiment, the present kit contains a setof primers (i.e., a forward primer and a reverse primer) (directed to aregion of the gene specific to a PME-1 biomarker and optionally ahybridization probe (directed to the same gene, albeit a differentregion)).

Kits provided herein may also include instructions, such as a packageinsert having instructions thereon, for using the reagents (e.g.,antibodies or primers) to quantify the protein expression level of mRNAexpression level of a particular endometrial cancer biomarker in abiological sample. Such instructions may be for using the primer pairsand/or the hybridization probes to specifically detect mRNA of aspecific gene (e.g., PME-1) in a biological sample. In another, theinstructions are directed to the use of antibodies (either monoclonal orpolyclonal) that recognize and bind to specific endometrial cancerbiomarker.

In another embodiment, the kit further comprises reagents used in thepreparation of the sample to be tested (e.g., lysis buffer). The kits ofthe invention also may further comprise one or more antibodies whichspecifically bind to PME-1 biomarker.

In one embodiment, the present invention provides a method of using theexpression levels of protein phosphatase methylesterase I (PPME1) andits gene product, protein methylesterase 1 (PME-1) as a biomarker for anincreased risk in endometrial cancer in humans. The present inventorshave established that PME-1 can serve as a biomarker for endometrialcancer, a novel finding that has not been previously recognized. Thepresent invention cures the long-felt needs in that it provides a methodfor determining whether a woman has an increased risk of endometrialcancer. The method involves detection of an increased level of PME-1, aswell as providing method of treatment through inhibition of PME-1, andkits containing the reagents and instruction necessary to employ themethod.

Epithelial to Mesenchymal Transition (EMT)

The present inventors discovered that over-expression of PME-1 inendometrial cells leads to increased endometrial cell migration andinvasive growth. The phenotype change is associated with activation ofERK and Akt cancer signaling pathways and increased phosphorylation ofβ-catenin, via inhibition of PP2A activity by PME-1. Over-expression ofPME-1 in endometrial cells decreases epithelial marker, E-cadherin, andincreased expression of mesenchymal markers (e.g., vimentin and Noggin).

The present inventors also discovered that depletion of PME-1 (by siRNAor shRNA) leads to a decrease in endometrial cell proliferation andinduces cell senescence.

Phenotypic change of endometrial cells induced by PME-1 is found to beassociated with EMT characteristics. EMT is involved in metastasis ofcancer. EMT is classified into three (3) types. Type 1 EMT involves thetransition of primordial epithelial cells into motile mesenchymal cells.This type is involved during embryonic development and organogenesis andis associated with the generation of diverse cell types. Type 2 EMTinvolves transition of secondary epithelial cells to resident tissuefibroblasts and is associated with wound healing, tissue regeneration,and organ fibrosis. Type 3 EMT occurs in carcinoma cells that haveformed solid tumors (such as endometrial cancer) and is associated withtheir transition to metastatic tumor cells that have the potential tomigrate through the bloodstream to distant sites. These three (3) typesof EMT represent distinct biological outcomes; however, the signals thatdelineate these subtypes are unclear.

EMT has multiple key hallmarks as evidenced by (1) loss of epithelialpolarity due to the loss of organized intercellular junctions, (2)cytoskeletal reorganization, and (3) acquisition of mesenchymalfeatures.

One key hallmark for EMT is the decrease in E-cadherin for epithelialcells (Engelsen, 2009). E-cadherin's normal function is to mediatecell-cell adhesion and thus maintain epithelial tissue integrity. A lossin E-cadherin alters the sequestration of associated cytoplasmicproteins, such as β-catenin. These features are thought to contribute toEMT and metastasis of endometrial cancer as the disease advances.

Another key hallmark for EMT is the increase of P-cadherin forepithelial cells. P-cadherin is a member of the classical cadherinfamily and is a transmembrane glycoprotein, normally found in theadherens junctions near the apical surface of a cell. Cadherins caninteract with β-catenin and are important for regulation of thecytoskeleton. An increase in P-cadherin, with concurrent decrease inE-cadherin levels, can suggest cells capable of migration or indicatemore aggressive cancers.

Another key hallmark for EMT is cytoskeletal reorganization.Specifically, several cytoplasmic proteins are used as markers for EMT.Vimentin, an intermediate filament protein present in most mesenchymalcells, is responsible for the strength and integrity of cells and itsmovements. β-catenin is an adhesion plaque protein that plays a dualrole during EMT. In epithelium, β-catenin is located in the cytoplasmand may be bound to E-cadherin. During EMT, β-catenin may translocateinto the nucleus and functions as a transcriptional activator togetherwith T cell factor (TCF/LEF) complex to regulate the expression of genesassociated with EMT. Nuclear accumulation of β-catenin has been detectedin cells undergoing EMT in embryonic development, fibrosis, and cancerand has been used as a biomarker for all three types of EMT.

In another aspect, the present invention provides RNAi knock-down ofPME-1 decrease epithelial to mesenchymal transition. There is a directcorrelation between the levels of PME-1 and the PP2A activity. Thepresent findings indicate that PME-1 is essential in regulation theprocess of epithelial to mesenchymal transition, as evidenced by siRNAtargeted against PME-1 that inhibits the transition process. Thus, thepresent invention provides a means to attenuate the endometrial cancerdevelopment.

The present invention therefore provides a therapeutic strategy of usingRNAi targeted against PME-1 as a treatment for endometrial cancer inwomen. For purposes of this application, RNAi is intended to encompassboth siRNA and shRNA. siRNA and shRNA targeted against PME-1 mRNAfunction equivalents to reduce mRNA of PME-1 mRNA. The present inventorsprovided the first elucidation for the regulation of the epithelial tomesenchymal transition process by PME-1 in female endometrial cancer.The disclosed data find support in human PME-1 gene may affect PP2A andthus regulate the development of endometrial carcinoma cells, probablyvia the epithelial to mesenchymal transition.

In one aspect, the present invention provides an isolated doublestranded short interfering ribonucleic acid (siRNA) molecule thatsilences expression of PME-1 mRNA. In another aspect, the presentinvention provides an isolated double stranded short interferingribonucleic acid (siRNA) molecule that silences expression of PME-1mRNA.

The mechanism of action of siRNA is understood by one skilled in theart. Interfering RNA (RNAi) generally refers to a single-stranded RNA ordouble-stranded RNA (dsRNA). The dsRNA is capable of targeting specificmessenger RNA (mRNA) and silencing (inhibiting) the expression of atarget gene. During the process, dsRNA is enzymatically processed intoshort-interfering RNA (siRNA) duplexes of 21 nucleotides in length. Theanti-sense strand of the siRNA duplex is then incorporated into acytoplasmic complex of proteins (RNA-induced silencing complex or RISC).The RISC complex containing the anti-sense siRNA strand also binds mRNAwhich has a sequence complementary to the anti-sense strand—allowingcomplementary base-pairing between the anti-sense siRNA strand and thesense mRNA molecule. The mRNA molecule is then specifically cleaved byan enzyme (RNase) associated with RISC resulting in specific genesilencing. For gene silencing or knock down (i.e., mRNA cleavage) tooccur, anti-sense RNA (i.e., siRNA) has to become incorporated into theRISC. This represents an efficient process that occurs in nucleatedcells during regulation of gene expression. When an anti-sense DNAmolecule is introduced into a cell, it targets specific mRNA throughbase-pairing of the anti-sense DNA molecule to its RNA target.

For purposes of this application, the anti-sense strand of the siRNA maycomprise a contiguous nucleotide sequence, where the base sequence ofthe anti-sense strand has sequence complementarity to the base sequenceof contiguous nucleotide sequence of corresponding length contained inthe mRNA sequence of the targeted mRNA (PME-1 mRNA). Complementaryincludes complete base-pairing match or a few base-pairing mismatches.

In one embodiment, the anti-sense strand of the siRNA molecule comprisesor consists of a sequence that is 100% complementary to the targetsequence or a portion thereof. In another embodiment, the anti-sensestrand of the siRNA molecule comprises or consists of a sequence that isat least about 90%, 95%, or 99% complementary to the target sequence ora portion thereof. For purposes of this application, the anti-sensestrand of the siRNA molecule comprises a sequence that specificallyhybridizes to the target sequence or a portion thereof so as to inhibitthe target mRNA expression. The present invention also encompassesanti-sense strand siRNAs that target the 3′UTR of the PME-1 RNA insofaras they possess similar activities to inhibit PME-1 mRNA expression.

Without wishing to be bound by a theory, siRNA-mediated RNA interferencemay involve two-steps: (i) an initiation step, and (ii) an effectorstep. In the first step, input siRNA is processed into small fragments,such as 21-23-nucleotide ‘guide sequences’. The guide RNAs can beincorporated into a protein-RNA complex which is capable of degradingmRNA, the nuclease complex, which has been called the RNA-inducedsilencing complex (RISC). The RISC complex acts in the second effectorstep to destroy mRNAs that are recognized by the guide RNAs throughbase-pairing interactions. siRNA involves the introduction by any meansof double stranded RNA into the cell which triggers events that causethe degradation of a target RNA. siRNA is a form of post-transcriptionalgene silencing. One of skilled in the art would understand thepreparation and utilization of siRNA molecules. (See, e.g., Hammond etal., Nature Rev Gen 2: 110-119 (2001); Sharp, Genes Dev 15: 485-490(2001), the disclosure of which are incorporated herein by reference intheir entireties).

Methods for preparing and isolating siRNA are known in the art (See,e.g., Smabrook et al., Molecular Cloning, A Laboratory Manual (2^(nd)Ed., 1989)), the disclosure of this is herein incorporated by referencein its entirety). In one embodiment, siRNA are chemically synthesized,using any of a variety of techniques known in the art, such as thosedescribed in Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); andWincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis of thesiRNA makes use of common nucleic acid protecting and coupling groups,such as dimethoxytrityl at the 5′-end and phosphoramidites at the3′-end. Suitable reagents for siRNA synthesis, methods for RNAdeprotection, and methods for RNA purification are known to those ofskill in the art. Small scale syntheses or large scale syntheses can beconducted using suitable synthesizer and protocols that are recognizedin the industry. Preferably, siRNA molecules are chemically synthesized.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousstrand separated by a cleavable linker that is subsequently cleaved toprovide separate strands that hybridize to form the siRNA duplex. Thetandem synthesis of siRNA can be readily adapted to both multi-well ormulti-plate synthesis platforms as well as large scale synthesisplatforms employing batch reactors, synthesis columns, and the like.Alternatively, siRNA molecules can be assembled from two distinctoligonucleotides, wherein one oligonucleotide comprises the sense strandand the other comprises the anti-sense strand of the siRNA. For example,each strand can be synthesized separately and joined together byhybridization or ligation following synthesis and/or de-protection. Incertain other instances, siRNA molecules can be synthesized as a singlecontinuous oligonucleotide fragment, where the self-complementary senseand anti-sense regions hybridize to form a siRNA duplex having hairpinsecondary structure.

In one embodiment, siRNA comprises a double stranded region of about 15to about 30 nucleotides in length. Preferably, siRNA has about 20-25nucleotides in length. The siRNA molecules of the present invention arecapable of silencing the expression of a target sequence in vitro and invivo.

In one embodiment, the siRNA comprises a hairpin loop structure. Inanother embodiment, the siRNA has an overhang on its 3′ or 5′ endsrelative to the target RNA that is to be cleaved. The overhang may be2-10 nucleotides long. In one embodiment, the siRNA does not have anoverhang (i.e., blunted).

In another embodiment, the siRNA molecule may contain one modifiednucleotide. In yet another embodiment, the siRNA may comprise one, two,three four or more modified nucleotides in the double-stranded region.Exemplary modified siRNA molecule includes, but not limited to, modifiednucleotides such as 2′-O-methyl(2′OMe) nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE)nucleotides, and the like. The preparation of modified siRNA is known byone skilled in the art.

The present inventors discovered PME-1 over-expression is correlatedwith the endometrial cancer development. An elevated PME-1 expression(both in protein and mRNA levels) is seen in tissues from endometrialcancer patients. Specifically, PME-1 is overexpressed in high stages andgrades in endometrial cancer.

The present observation that there is a correlation in the PME-1 proteinexpression in matched pair of human endometrial tumor samples furthersubstantiates the important role of PME-1. The present inventionprovides a therapeutic approach of employing siRNAs to block the PME-1expressions and thus attenuate the endometrial tumor development.

In one aspect, the present invention provides exemplary anti-sensestrand siRNAs that hybridize to the PME-1 mRNA so as to increasedegradation of PME-1 mRNA (and consequently PME-1 protein expression).In one embodiment, the present invention provides exemplary anti-sensestrand siRNA targeted against PME-1 (SEQ ID NOs: 2-3 and SEQ ID NOs:5-7) that hybridizes to PME-1 mRNA.

The present RNAi molecule targeting PME-1 can be used to down-regulateor inhibit the expression of PME-1. The PME-1 expression is inhibited atleast about 40%-100%.

RNAi may conveniently be delivered to a target cell through a number ofdirect delivery systems. For example, RNAi may be delivered viaelectroporation, lipofection, calcium phosphate precipitation, plasmids,viral vectors, viral nucleic acids, phage nucleic acids, phages,cosmids, or via transfer of genetic material in cells or carriers suchas cationic liposomes. In one embodiment, transfection of RNAi mayemploy viral vectors, chemical transfectants, or physico-mechanicalmethods such as electroporation and direct diffusion of DNA. The RNAidelivery methods are known in the art and readily adaptable for use.(See, e.g., Wolff, J. A., et al., Science, 247, 1465-1468, (1990); andWolff, J. A. Nature, 352, 815-818, (1991)).

In one aspect, the present invention provides a pharmaceuticalcomposition containing RNAi targeted against PME-1 for the treatment ofendometrial cancer. The pharmaceutical composition comprises the RNAi astherapeutic agents for inhibiting PME-1 gene activity and apharmaceutical acceptable carrier. Pharmaceutically acceptable carriersinclude, but are not limited to, excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, toaid absorption in the gastrointestinal tract.

Pharmaceutical compositions containing RNAi may be administered to amammal in vivo to treat cancer. In one embodiment, the pharmaceuticalformulation includes a dosage suitable for oral administration. Inanother embodiment, the pharmaceutical formulation is designed to suitvarious means for RNAi administration. Exemplary means include uptake ofnaked RNAi, liposome fusion, intramuscular injection via a gene gun,endocytosis and the like. These administration means are well known inthe art.

The present invention provides a means for attenuating (i.e.,inhibiting) the PME-1 gene using RNAi targeting against PME-1. Thepresent invention provides a method of reducing epithelial tomesenchymal transition in endometrial cancer cells by reducing PME-1expression levels. Specifically, the present invention provides a methodof using RNAi targeted against PME-1 in attenuating the PME-1 expressionin endometrial cancer. The use of RNAi to attenuate PME-1 represents aprognostic tool in treating human endometrial carcinoma.

In one embodiment, the RNAi is administered to a human with atherapeutic effective amount of RNAi targeting PME-1. The specificamount that is therapeutically effective can be readily determined bymonitoring the PME-1 mRNA levels Inhibition of PME-1 mRNAs isconveniently achieved by using qRT-PCR, Northern blot analysis and othertechniques known to those of skill in the art such as dot blots, in situhybridization, and the like. The inhibition level is comparing thetarget gene expression to the control. A detectable inhibition can beabout 40%-100%. Preferably, the % inhibition may be 80%, 90% or 100%.The therapeutic effective amount may be determined by ordinary medicalpractitioner, and may vary depending on factors known in the art, suchas the patient's history and age, the stage of pathological processesmediated by PME-1 expression.

The following examples are provided to further illustrate variouspreferred embodiments and techniques of the invention. It should beunderstood, however, that these examples do not limit the scope of theinvention described in the claims. Many variations and modifications areintended to be encompassed within the spirit and scope of the invention.

EXPERIMENTAL STUDIES Example 1 PME-1 Expression in Endometrial CellLines

In these initial studies, we sought to determine the expression levelsof PME-1 in various human endometrial cell lines (EC). We examined theexpression level of PME-1 protein using Western Blot analysis (See,Materials and Methods).

As shown in FIGS. 1 and 2, the more aggressive/metastatic EC cell lines(RL95-2, Ishikawa, and ECC-1) were found to express more abundant PME-1protein as compared to the less aggressive/metastatic EC line (KLE) andthe immortalized endocervical cell line, End1. GAPDH was used as aloading control in these studies. These data suggest that expression ofPME-1 protein levels correlates with the degree of aggressiveness of EC;that is the more aggressive/metastatic EC, the higher the expression ofPME-1 protein levels.

Example 2 Western Blot Analysis of PME-1 Protein Expression in TissueSamples from Patients with Endometrial Adenocarcinoma

After establishing that the degree of aggressiveness and metastasis inEC cell lines indeed correlates with an increased level of PME-1protein, we next determined whether the same trend might occur in tissuesamples from patients.

We obtained 30 matched pairs of tissue samples harvested from patientswith endometrial adenocarcinoma. The details of the relevant patientdata are provided in Table 1. We used Western Blot analysis to monitorthe expression of PME-1 protein levels in these tissue samples frompatients (FIG. 3).

Representative Western blots are displayed for several stage I ECpatient samples. All stage II and stage III tissue samples purchased arealso shown in FIG. 3. The top left panel shows stage IB patient samples(patient #6241, 6247, 6248, 6262) except for #6262, which is a grade 1tumor. The top right panel shows three (3) stage IC in which only onesample (patient #6336) displays an increase in expression of PME-1protein in the tumor sample (T) versus the NAT sample (N). Three (3) outof seven (7) stage I patient EC samples show only a detectable PME-1protein level in the tumor sample (patient #6241, 6247, 6336); however,one patient sample (#6247) expressed PME-1 protein in the NAT sample.The bottom left panel displays both Stage II samples (patient #6313,6333) and there is a slight increase in PME-1 protein for both patients.

Importantly, PME-1 protein was found to increase in all stage IIIpatient tumor samples as compared to their corresponding NAT samples(patient #6310, 6308, 6275, 6261).

As shown in FIG. 4, PME-1 levels were assessed in patient samplesaccording to FIGO grade. In general, PME-1 expression increased in tumortissue (T) as compared to NAT samples. For purposes of this application,the comparison in our clinical study involves comparing tumor tissuesamples in patients suffering from endometrial cancer relative to thenormal adjacent tissue samples from the same individual. For practicalpurposes, our clinical assay can be performed using normal tissuesobtained from normal women (free of endometrial cancer) instead of thenormal adjacent tissues.

Altogether, the present data demonstrate that PME-1 protein levelsincrease as grade and/or stage increases, suggesting that PME-1 levelscorrelate with the aggressiveness of the endometrial cancer. BecausePME-1 protein levels were found to increase in all four stage III ECpatients and the increase was dramatic as compared to the NAT samples,we conclude that there is a correlation in PME-1 levels and highgrade/stage of EC tumors.

Example 3 Immunohistochemistry Analysis

We confirmed the PME-1 protein expression data by evaluating theimmunohistochemistry of the tissue samples from EC patients. Thisimmunohistochemistry study provides the basis for our observation of anincreased PME-1 expression level in the context of tissue morphology.

We performed H&E staining on the tissue samples (FIG. 5i -iv) andco-stained tissue samples with anti-PME-1 antibodies (FIG. 5v -viii).Normal tissue showed regular duct formation in the endometrium withlight PME-1 expression around ducts within epithelial cells, asillustrated by the black arrows (FIG. 5i, v ). In stage I EC, ductsbecame irregular and PME-1 localization becomes more punctuate andlocalized, as illustrated by the black arrows (FIG. 5 ii, vi). Stage IIEC was even less organized with increased PME-1 staining and more foci,as illustrated by the black arrows (FIG. 5 iii, vii).

In stage III EC, PME-1 staining was darker and appeared to be limited toindividual cells, marked by black arrows (FIG. 5 iv, viii), which wasconsistent with the hypothesis that the cells were migrating through thetumor stroma due to their elongated morphology. The data support ourWestern blot analysis that PME-1 expression levels increased withincreased stages. The visualization of migrating cells darkly stainedfor PME-1 in Stage III tumors suggest that PME-1 expression is increasedin cells that have undergone epithelial-to-mesenchymal transition (EMT).

Samples were analyzed by grade as well (FIG. 6). Patient samples weresectioned, H&E stained, and graded according to the InternationalFederation of Gynecology and Obstetrics (FIGO) grading system forendometrial cancer. In the top panel, H&E staining was conducted toexemplify the differences in endometrial cancer grade. Grade 1 tumorsare well-differentiated cancers with clear cellular boundaries andnormal cell morphology. Grade 2 tumors are moderately differentiatedwith abnormal cell morphology. Grade 3 tumors are poorly differentiatedexhibiting loss of clearly defined boundaries and highly abnormal cellmorphology.

The bottom panels depict representative immunohistochemistry slides inwhich sections were H&E stained and were immunostained with antibodiesspecific for PME-1. PME-1 staining intensity was then graded accordingto the following scale: 0 indicates negative cytoplasmic PME-1 stainingor faint staining observed in <50% of the cells; 1+ indicates weaklypositive cytoplasmic PME-1 staining in >50% of tumor cells ormoderate/strong staining in <50% of tumor cells; 2+ indicates stronglypositive staining with moderate/strong cytoplasmic staining observedin >50% of the tumor cells.

The table in FIG. 6 summarizes the results for PME-1 staining intensity.Of the 9 FIGO grade 1 samples examined, 55% were 1+ and 45% were 2+,whereas of the 7 FIGO grade 2 samples examined, 43% were 1+ and 57% were2+. All FIGO grade 1 and grade 2 samples exhibited weak to strongstaining for PME-1. Only three FIGO grade 3 samples were examined by IHCfor PME-1 and no trend was determined. PME-1 immunopositivity wasdetected in 18 of 19 samples (−95%) tested by IHC (as shown in FIG. 6 inthe table), suggesting that PME-1 strongly correlates with disease. Anincrease in PME-1 2+ staining was observed in grade 2 compared to grade1 patient samples (57% versus 44%), suggesting that PME-1 levelsincrease as the tumor grade increases from 1 to 2.

Example 4 PME-1 mRNA Expression

So far, our data indicate a correlation between increased stages of ECpatient samples with increased expression level of PME-1 protein. Inthis series of studies, we examined if PPME1 mRNA expression levels wereincreased in patient samples.

We used quantitative real-time PCR to determine the mRNA levels ofPPME-1 in the EC patient tissue samples. Total RNA was extracted fromtissue samples of matched pair, tumor (T) and normal adjacent tissue(NAT, N). Patient data is presented in Table 1. The level of PPME1, thegene that codes for PME-1, was determined by qPCR using 18S to normalizePME-1 expression. All samples were then normalized to PME-1 expressionin RL95-2 cells, which was set to a value of 1.

The mean expression of PPME1 was >20-fold higher in tumor samples ascompared to that of normal samples (FIG. 7A). Statistical significancewas determined via the Mann-Whitney non-parametric statistics test.Importantly, ROC analysis (FIG. 7B) demonstrates that detection of PPME1levels may be a beneficial diagnostic marker, as the area under thecurve (AUC) score is >0.9, the sensitivity of the assay is 80.77% andthe specificity is 96.15%, when the likelihood ratio is 21, suggestingthat PME-1 levels may be predictive of EC. When we compared the ratio ofPPME1 expression within matched pairs of the patient samples (i.e. thelevel of PPME1 in tumor versus the level of PPME1 in the normal adjacenttissue from the same patient), we found that eleven (11) out of thirteen(13) FIGO grade 1, ten (10) out of twelve (12) FIGO grade 2, and three(3) out of five (5) FIGO grade 3 tumors had very high levels of PPME1 inthe tumor samples (Table 1). In sum, these data suggest that PME-1 mayserve as a useful biomarker for determining more aggressive EC risk formetastasis as higher levels correlate with more aggressive cancers.

Example 5 Effects of siRNA/shRNA Targeted Against PME-1

To confirm our findings that PME-1 levels may be predictive ofaggressiveness and metastasis ability in EC, we used the RL95-2 EC cellline, which we transfected with siRNA or shRNA against PME-1. The RL95-2EC cells were chosen because they represent a highly metastaticendometrial adenocarcinoma cell line.

We prepared RL95-2 cell lines expressing empty vector (control),over-expressing PME-1 (+PME-1), or expressing shRNA against PPME1, thegene that codes for PME-1 protein (−PME-1). Using these cells, weconfirmed the appropriate level of PME-1 via real-time RT-PCR (data notshown) and Western blot analysis (FIG. 8). GAPDH was used as a loadingcontrol.

We also prepared two different shRNAs targeted against PME-1. BothshRNA#1 and shRNA#2 showed significantly decreased PME-1 levels viaWestern blot analysis (SEQ ID NOs: 2 or 3). For all subsequent studies,shRNA #2 was used since it showed the best depletion of PME-1 protein inRL95-2 cells.

Example 6 siRNA Targeted Against PME-1 Increases PP2A Activity

Because PME-1 is a known inhibitor of PP2A, we confirmed in this studythat depletion of PME-1 by siRNA led to an increase in PP2A activity incells. To do so, we treated RL95-2 cells with either non-targeting siRNA(Control) or siRNA against PPME1. PP2A activity was monitored using aphosphatase assay (R&D Systems Duo IC Set phosphatase assay kit). In thephosphatase assay, we used 500 μg of whole cell extract and followed themanufacturer's recommendations.

We found that shRNA targeted against the PPME1 gene led to a 30-foldincrease in PP2A activity, confirming that siRNA against PPME1 regulatesPP2A activity (FIG. 9A). These data indicate that inhibition of PME-1significantly increases PP2A activity. Conversely, we found thatover-expression of PPME1 led to a significant 90% decrease in PP2Aactivity (FIG. 9B). These data indicate that increased PME-1 activitydecreases PP2A activity. We speculate that increase (reactivation) ofPP2A activity in cancer may serve as a beneficial therapeutic means toslow tumor growth and metastasis.

Example 7 Foci Forming Assay

In this series of study, we tested the hypothesis that PME-1 may affecttumor cell growth pathways. Specifically, we tested that inhibition ofPME-1 would lead to a decrease in cell growth or proliferation.

A foci forming assay was used in this study to monitor cell growth.Equal amounts of cells were plated and they were allowed to grow for adetermined period of time (see Materials and Methods section fordetails). The foci that were formed were then stained with the crystalviolet dye. Cells were washed 3× with PBS prior to visualization.Numbers of foci were counted and results were averaged among differentexperiments.

As shown in FIG. 10, there was a 50% increase in foci when PME-1 wasover-expressed. In contrast, a 75% decrease in foci was observed whenPME-1 was depleted as compared to control cells (FIG. 10A).

Example 8 Foci Forming Assay—Confirmation

To determine if the loss of active PME-1 accounted for the observeddecrease in foci formation, we first treated RL95-2 cells with siRNAagainst the 3′-UTR of PPME1 to decrease endogenous levels of PME-1. Thenwe overexpressed either an empty vector or a catalytically inactive formof PME-1 (S156A—in which the nucleophilic serine residue is mutated toan alanine) in the RL95-2 cells (FIG. 10B). The S156A PME-1 mutant isnot capable of demethylating its target, PP2A.

When PME-1 was depleted from the treatment with siRNA against the 3′-UTRof the PPME1 gene followed by expression with an empty vector, there wasa significant 40% decrease in foci formed when compared to the controlsiRNA treatment with empty vector expression. When we depleted theendogenous PME-1 with the 3′-UTR siRNA and over-expressed the S156Amutant form of PME-1, there was still a 40% reduction in foci formation.

When cells were treated with 3′-UTR siRNA and wild type PME-1 was addedback in a vector, foci formation was rescued and returned to a higherlevel than in the control siRNA+Empty vector sample (data not shown).These data suggest that active PME-1 is required for the maintenance offoci formation and increased tumor cell proliferation.

Example 9 BrDU Incorporation Assay

To confirm our foci formation data that altering PME-1 levels inendometrial cancer cells affect their rate of cell proliferation, weconducted a BrDU incorporation assay. BrDU is incorporated into newlysynthesized DNA and can be detected with antibodies; thus, increasedBrDU incorporation correlates with an increased level of proliferation.

We found that RL95-2 cells over-expressing PME-1 (+PME-1) exhibited asignificant 40% increase in BrDU incorporation compared to empty vectorcontrol (FIG. 11), whereas depleting cells of PME-1 (−PME-1) with shRNAled to a significant 40% decrease in BrDU incorporation compared to ascrambled shRNA control (FIG. 11). Each control was set to a value of100% incorporation. These data further suggest that PME-1 promotes cellproliferation.

Example 10 Immunofluorescence Assay

To confirm our foci formation and BrDU incorporation data that suggestthat altering PME-1 levels in endometrial cancer cells affect their rateof cell proliferation, we conducted an immunofluorescence assay.Endometrial cancer cells were stained for phosphorylated histone H3 onserine 10, a marker of mitotic cells, followed by counterstaining withDAPI (FIG. 12) to identify all cells.

We found that 2.3-fold more +PME-1 cells were actively proliferating andthat there was ˜30% decreased proliferation in −PME-1 cells compared tocontrol cells. Taken together, these data suggest that PME-1 promotescell proliferation.

Example 11 TUNEL Assay

Our data show that a decrease in levels of PME-1 in RL95-2 cells,whether through siRNA or shRNA targeting of PPME1 message, led to adecrease in cell growth. A decrease in cell growth might be due to cellsentering programmed cell death, or apoptosis, or cell senescence, astate in which a cell exits the cell cycle.

To test for the involvement of apoptosis, we used siRNA against PME-1 orcontrol siRNA and conducted an immunofluorescence assay called a TUNELassay. In this assay, all cells were stained blue with the nuclearstain, DAPI, and cells that were undergoing apoptosis would stain greenwith the addition of fluorescein, which bound to fragmented DNA.Fragmented DNA is a hallmark of apoptotic cells. We found that thedecrease in cell proliferation of cells depleted for PME-1 was not dueto apoptosis, since PPME1 siRNA treatment did not lead to detection ofapoptotic cells (FIG. 13A). A positive control was included in the assayto determine that the assay conditions were optimal. In the positivecontrol, RL95-2 cells were treated with DNase. In sum, the TUNEL assaydemonstrated that loss of PME-1 does not cause apoptosis in EC cells.

Example 12 Senescence Assay

We next analyzed cell lysates taken from RL95-2 cells that expressedeither empty vector (Control), overexpressed PME-1 (+PME-1) or expressedshRNA against PME-1 (−PME-1) for DcR2 protein. DcR2 protein is a markerof senescent cells and the expression level of DcR2 protein was shown toincrease when cells are senescing.

Western analysis demonstrates that loss of PME-1 correlates with anincrease in the senescent marker protein, DcR2, suggesting thatinhibition of PME-1 activity pushes cells towards senescence (FIG. 13B).In sum, these data support our hypothesis that inhibition of PME-1increases PP2A activity and thus decreases cell proliferation in the ECcell line. We also show that loss of PME-1 activity leads to a decreasein cell growth that is not caused by apoptosis, but by cell senescence.

Example 13 PME-1 Expression Correlates with EMT

So far, we showed that PME-1 promotes cell proliferation (FIGS. 10, 11,12) and that PME-1 expression increases in late stage/high grade EC(FIGS. 3, 4, 5, 6, Table 1). In the immunohistochemistry study, weobserved that PME-1 staining in stage III EC occurred in cells thatappear to be migrating through the tumor stroma (FIG. 3 viii). Based onthese results we hypothesized that PME-1 plays a role inepithelial-to-mesenchymal transition (EMT).

In this study, we examined if PME-1 was expressed in cells that alsoexpressed E-cadherin, a marker of epithelial cells, or P-cadherin, amarker of aggressive or mesenchymal endometrial cancer cells. Tissuesamples were purchased from Proteogenex and were analyzed by fluorescentmicroscopy (FIG. 14). In this Figure, PME-1 was shown in green, andP-cadherin (FIG. 14A) and E-cadherin (FIG. 14B) were displayed in red.Merged figures showed co-expression of the proteins in yellow and aremarked with white arrows.

We found that both grade 1 (patient 06313) and grade 3 (patient 06294),PME-1 and P-cadherin expression was increased compared to correspondingNAT samples (FIG. 14A). In grade 1 samples, more cells expressed PME-1and P-cadherin, but in grade 3 samples, we noted single cells stainingbrightly for both proteins (arrows in merge). We then examined PME-1 andE-cadherin expression in grade 1 and grade 3 tumors. There is overlap inPME-1 and E-cadherin expression in the grade 1 sample (FIG. 14B, arrowsin merge), but in grade 3 EC, PME-1 is not co-expressed with E-cadherin,suggesting that the cells expressing PME-1 in grade 3 tumors are nolonger epithelial but mesenchymal in nature.

Example 14 Colony Forming Assay

To determine if PME-1 contributes to the more invasive and metastaticphenotypes, we performed a colony forming assay in matrigel.RL95-2+PME-1 cells formed 50% more colonies than control cells, whereas−PME-1 cells exhibited a 50% decrease in colony formation (FIG. 15).

Importantly, the colonies that were formed by +PME-1 cells were lessspherical (FIG. 16, −TGF-β) as compared to the control cells andexhibited rough edges (arrows), suggesting migration of the cells fromthe cyst. Moreover, −PME-1 cells produced fewer and smaller colonies ascompared to the control cells, suggesting that loss of PME-1 decreasesinvasive growth phenotypes in EC. Upon addition of TGF-β, which inducesEMT, we found that there is an overall decrease in colony number;however, cells over-expressing PME-1 (+PME-1) formed large and highlyirregular colonies compared to control and −PME-1 cells (FIG. 16,+TGF-β)

Example 15 Raf/MEK/ERK Signaling Pathways

Another signaling event common in many proliferating cells is theactivation of the Raf/MEK/ERK and Akt signaling pathways. Continuousactivation of these pathways is believed to promote EMT. Activation ofthe Raf/MEK/ERK pathway can be detected by examining the phosphorylationstatus of ERK. Furthermore, phosphorylation of Akt on S473 and/or T308represents activation of the Akt signaling pathway.

In this study, we show via immunofluorescence that we successfullyover-expressed PME-1 in RL95-2 cells (compare FIG. 17Aii to FIG. 17Ai).Importantly, we found that the increase in PME-1 levels correlates withan increase in phosphorylated ERK (FIG. 17Bii compared to FIG. 17Bi)while the levels of total ERK remain unchanged (FIG. 13Biii-iv).

Western analysis confirmed these results in RL95-2 cells; when PME-1 wasover-expressed, there was a significant increase in phosphorylated ERK,while total ERK levels remain unchanged (FIG. 18A). When PME-1 wasdepleted with shRNA, there was a decrease in phospho-ERK compared tocontrol cells.

Treatment of cells with UO126 (an upstream inhibitor of ERKphosphorylation) demonstrates the specificity of the phospho-ERKantibody. In sum, the data support our hypothesis that increased levelsof PME-1 promote cell proliferation through activation of theRaf/MEK/ERK signaling pathway.

Example 16 Increased Phosphorylation of Akt

We observed a similar trend for the Akt signaling pathway. There was anincrease in phosphorylation of Akt on threonine 308 (T308) when PME-1was over-expressed compared to control. There was a decrease inphosphorylation of Akt when PME-1 was knocked down (FIG. 18B). PME-1over-expression specifically increases the phosphorylation of Akt onT308, but not serine 473 (S473). Importantly, when cells were incubatedwith upstream inhibitors of Akt phosphorylation (LY294002), there was adecrease in the phospho-forms (FIGS. 18A=−B), as expected. Takentogether, these data implicate PME-1 to be a positive regulator of theERK and Akt cancer signaling pathways in EC cells. Activation of boththe ERK and Akt signaling pathways has been demonstrated to increasecell proliferation in cancer cells. Importantly, we found that byinhibiting PME-1 activity through decreasing the level of PME-1 protein(via either siRNA or shRNA) there was a decrease in ERK and Aktphosphorylation, which is indicative of decreased cell growth.

Example 17 β-Catenin Phosphorylation

PP2A has been shown to negatively regulate cell migration through thedephosphorylation of β-catenin, protecting it from ubiquitylation anddegradation. Because PME-1 has been shown to decrease the association ofthe B/55α regulatory subunit to the catalytic subunit of PP2A andincreased PME-1 seems to promote cell proliferation and EMT, we asked ifover-expression of PME-1 in EC cells promoted β-catenin phosphorylationand stabilization.

We performed Western blot analysis in RL95-2 cells that were expressingempty vector (Control), over-expressing PME-1 (+PME-1) or expressingshRNA against PME-1 (−PME-1) to examine the protein stability ofβ-catenin (FIG. 19A). We observed a decrease in total β-catenin proteinin cells that were over-expressing PME-1. This observation is correlatedwith the predicted ubiquitylation of β-catenin and its degradation.Conversely, a depletion of PME-1 led to stabilization of total β-catenin(FIG. 19A).

In another series of study, we performed a similar assay in which cellswere treated with 10 μM MG-132 prior to protein extraction. MG-132 is aproteasome inhibitor and therefore allows for the accumulation ofphosphorylated β-catenin, which is normally quickly degraded, to bedetected by western analysis. We found that over-expression of PME-1increased β-catenin phosphorylation on S45 as well as the secondaryphosphorylation sites, S33, S37, and T41 (FIG. 19B) when cells weretreated with MG-132. Conversely, we found that decreasing PME-1 levelswith shRNA did not lead to an increase in phosphorylated β-catenin whencompared to control cells, as expected. These data support ourhypothesis that PME-1 regulates β-catenin stability through inhibitionof B/55α-dependent PP2A.

Example 18 Expression of EMT Markers

We determined if manipulating PME-1 levels in RL95-2 cells alteredexpression of EMT markers, such as the epithelial marker, E-cadherin,and mesenchymal markers, vimentin and noggin.

In this study, we transfected RL95-2 cells to express empty vector(Control), over-expressing PME-1 (+PME-1) or expressing shRNA againstPME-1 (−PME-1) followed by serum starvation for 24 hours. Afterwards,either vehicle or TGF-β treatment was performed at the end of the 24hours. TGF-β was used to induce cells to initiate the EMT program. Geneexpression analysis was conducted using quantitative real-time PCR.

We discovered that over-expression of PME-1 led to a decrease inE-cadherin expression (FIG. 20A) and a concomitant ˜2.5-fold increase invimentin expression (FIG. 20B), which is consistent with cellsundergoing EMT. With TGF-β treatment, the effects of PME-1over-expression on vimentin levels were more dramatic.

We also noted an increase in Noggin expression (FIG. 20C) when PME-1 wasover-expressed compared to control cells. A ˜2-fold increase in PME-1expression was detected in control cells and +PME-1 cells upon additionof TGF-β (FIG. 20D), suggesting that PME-1 is induced by TGF-βstimulation. These data suggest that an increased level of PME-1 inducesendometrial cells to undergo EMT.

The Wnt signaling pathway is a well-characterized pathway thatcontributes to increased cell proliferation, especially EMT (Koval, DrugDisc. Today 17: 1316 (2012). Wnt proteins bind to Frizzled receptors,which are GPCR protein receptors, which signal through β-catenin toincrease the expression of genes involved in cell proliferation and EMT(King et al., Cancer Signalling 24:846-851 (2012). We asked if alteringthe levels of PME-1 affected the expression levels of Wnt signalinginhibitor, such as secreted frizzled-like protein (SFRP1), or Wntsignaling activators, such as Wnt3 and Wnt3A.

Using RL95-2 endometrial cancer cells, we overexpressed PME-1 (FIG. 21A)or depleted PME-1 with shRNA (FIG. 21B) and confirmed that PME-1 wasappropriately expressed. We then examine the relative expression of theWnt signaling inhibitor, SFRP1, and found that when PME-1 wasover-expressed, SFRP1 expression was significantly decreased (FIG. 21C),and when PME-1 was depleted, SFRP1 expression was significantlyincreased (FIG. 21D). Conversely, when PME-1 was over-expressed, therewas a significant increase in expression of Wnt3 and Wnt3A, which areactivators of this pathway (FIG. 21E), and that when PME-1 was depleted,Wnt3 and Wnt3A expression were significantly decreased (FIG. 21F). Takentogether, these data suggest that PME-1 promotes metastasis throughactivation of the Wnt signaling pathway.

Example 19 PME-1 Binds PP4 in Addition to PP2A

(a) PME-1 Binds to PP2A Via Ppp2ca

We next evaluated the binding affinity of PME-1 to PP2A in endometrialcancer cells. We chose to examine the PME-1 binding domain on PP2A. Thecatalytic subunit of PP2A is coded by two genes (namely, PPP2CA andPPP2CB), each produces a protein respectively (namely, Ppp2ca andPpp2cb, also known as α and β isoforms of PP2A, respectively). Ppp2caand Ppp2cb proteins are 83% identical. At the C-terminus of Ppp2ca andPpp2cb, they are 100% identical.

To evaluate the binding domain between PME-1 and PP2A, we transfectedECC-1 cells with either (i) empty FLAG vector, (ii) FLAG-PME-1, or (iii)FLAG PME-1 S156A. When the serine (S) residue at the 156 position onPME-1 is mutated to an alanine (A) residue, the PME-1 S156A mutant iscatalytically inactive (i.e., no long be able to demethylate PP2A).Following the transfection, we immunoprecipitated using FLAG resin andperformed a Western blot on the eluants using various antibodies (i.e.,anti-FLAG and anti-Ppp2ca) to determine the binding between PME-1 andPpp2ca or Ppp2cb.

As shown in FIG. 22A, PME-1 binds (albeit a less degree) to Ppp2ca. Alonger exposure (i.e., 2 min, lighter exposure was 10 sec) shows thePME-1 binding. FIG. 22A also show that PME-1 S156A binds to Ppp2ca morestrongly. We noted that PME-1 also binds equally well to Ppp2cb (datanot shown). We concluded that PME-1 binds to the catalytic subunit ofPP2A (i.e., Ppp2ca or Ppp2cb) transiently. The binding of PME-1 S156A isstronger due to its inability to demethylate its substrate.

(b) PME-1 Binds to Another Protein Phosphatase (PP4) Besides PP2A

There are other protein phosphatases in the same family as PP2A in cellsthat share some similarity to PP2A. In this experiment, we examined ifPME-1 binding to Ppp2ca is specific. We chose to examine phosphatase 4(PP4) or phosphatase 6 (PP6) since the C-terminal tail of the catalyticsubunit of PP2A (i.e., Ppp2ca) is TPDYFL (SEQ ID NO: 9) (the substratefor PME-1 demethylation) is similar to that of protein phosphatase 4(i.e., Ppp4c) (VADYFL) (SEQ ID NO: 10) and phosphatase 6 (i.e., Ppp6c),(TTPYFL) (SEQ ID NO: 11), respectively. Specifically, we examined ifPME-1 may bind and demethylate PP4 or PP6. PP6 does not seem to beregulated by PME-1 in EC cells since we could not detect binding betweenPME-1 or PME-1 S156A with Ppp6c.

In this experiment, we transfected ECC-1 cells with either (i) emptyFLAG vector, (ii) FLAG-PME-1, or (iii) FLAG PME-1 S156A. Following thetransfection, we immunoprecipitated using FLAG resin and performed aWestern blot on the eluants using (i) anti-FLAG, (ii) anti-Ppp4c, and(iii) anti-Ppp6c. As shown in FIG. 22B, PME-1 binds to Ppp4c, but notPpp6c. We concluded that PME-1 also binds to other phosphatase such asPpp4c, and predicted that PME-1 may demethylate PP4. Note that Ppp4cshares 68% identity with Ppp2ca/b and Ppp6c shares 70% identity withPpp2ca/b.

Together, this data provide that PME-1 binds to Ppp2ca or Ppp4ctransiently and causes Ppp2ca/Ppp4c to undergo demethylation We foundthat the binding of PME-1 to Ppp2ca/Ppp4c is independent of itscatalytic activity (i.e., S156A) and that PP6 is not regulated by PME-1in EC cells.

(c) PME-1 Binds to PP2A with a Stronger Affinity than that of PP4

In this series of study, we co-transfected HEK293T cells with (i) emptyV5 vector, (ii) V5-PME-1, or (iii) V5-PME-1 S156A in combination with(i) empty FLAG vector, (ii) FLAG-Ppp2ca, or (iii) FLAG-Ppp4c (FIG. 23).We determined if PME-1 has a higher affinity for PP2A than that of PP4.

We performed a Western blot analysis of the input samples, which areprotein lysate samples that are removed and saved for western analysisto determine the starting amount of protein material prior to completingimmunoprecipitation. We observed that Ppp2ca and Ppp4c (FLAG antibody)were expressed at equal levels and V5-PME-1 and V5-PME-1 S156A wereexpressed at equal levels (V5 antibody, FIG. 23A). We then performed aFLAG elution Western blot analysis. We observed that both FLAG-Ppp2caand FLAG-Ppp4c were immunoprecipitated and expressed at equal levels. Wealso observed that V5-PME-1 binds at a lower affinity (likely due to atransient interaction) than V5-PME-1 S156A (FIG. 23B). These datasuggest that PME-1 has a stronger affinity for binding to PP2A than PP4,because the α-V5 band is stronger for V5-PME-1 S156A when FLAG-Ppp2ca isimmunoprecipitated, as compared to that when FLAG-Ppp4c isimmunoprecipitated.

(d) Gene Silencing of PME-1 with shPME-1 Treatment Causes Foci Formation

In this series of study, we examined the effects of PME-1 inhibition oncell growth when PP2A, PP4, or PP6 were over-expressed. While PP2A is atumor suppressor, PP4 promotes tumorigenic phenotypes. Because weidentified that PME-1 is able to bind both PP2A and PP4, we tested hereif decreased PME-1 levels was beneficial, when PP4 tumor promotingactivity was increased. We performed foci formation studies when cellswere over-expressing PP2A, PP4, or PP6 and compared the effect of PME-1in counter-acting the tumor promoting effects mediated by PP2A or PP4.

We performed a foci formation analysis by transfecting (i) empty FLAGvector, (ii) FLAG-PPP2CA, (iii) FLAG-PPP4C, or (iv) FLAG-PPP6C intoECC-1 cells that stably expressed either (i) shSCR (non-targeting shRNA,black bars) or (ii) shPPME1 (depletes PME-1 levels, white bars) (FIG.24).

As shown in FIG. 24, inhibition of PME-1 leads to a significant decreasein foci formation (i.e., decrease in cell proliferation) as compared tothe control (Empty, black bar). In cells transfected with PP2A, PP4 orPP6, inhibition of PME-1 similarly leads to a significant decrease infoci formation (i.e., decrease in cell proliferation). Altogether, thesedata suggest that gene silencing PME-1 is a valid target for cancertherapy.

Example 20 Chemical Inhibition of PME-1 Leads to Decreased CancerousPhenotypes

(a) PME-1 Inhibitors Directly Bind PME-1

There are several commercially available covalent PME-1 inhibitors thatwe have analyzed as potential endometrial cancer therapeutics. Toconfirm that the inhibitors directly bind PME-1, we completed a thermalmelt assay. One micromole (1 μmole) of recombinant PME-1_des3, of whichthe last 3 amino acids are deleted, were pre-incubated with SYBR orangein the presence of 50 μM ABL-127 (a potent covalent PME-1 inhibitor,squares) or 0.05% DMSO (vehicle, circles) for 10 minutes (FIG. 25) at37° C. We then performed a thermal gradient, increasing the temperatureby 2° C. each minute from 37° C. to 95° C. and fluorescent signal wasdetected at 492-601 nm on a thermal cycler. The increase in Tm from ˜55°C. (PME-1+DMSO, circles) to ˜65° C. (PME-1+ABL-127, squares) indicatedthermal stabilization of the PME-1 protein via direct binding of theABL-127 inhibitor (FIG. 25).

(b) PME-1 Inhibition Leads to Decreased Cell Proliferation

In order to determine if compound-based inhibition of PME-1 decreasedcell proliferation as we showed with RNAi-based depletion of PME-1, wecompleted a foci formation assay, as before (FIG. 10), using thecovalent inhibitors of PME-1, ABL-127 and AMZ-30. This study wascompleted in Ishikawa endometrial cancer cells. As shown in FIG. 26,DMSO served as the vehicle control (set to 100%) and Ishikawa cells weretreated with 50 nM ABL-127 or 25 μM AMZ-30 every 2-3 days for a total of10 days. shSCR- and shPPME1-treated cells were used for comparison andwere normalized to untreated treated cells (not shown) set to 100%.

We found that treating Ishikawa cells with the covalent PME-1inhibitors, ABL-127 and AMZ-30 led to ˜50% decrease in the amount offoci that were formed, indicating significant decreases in cellproliferation (FIG. 26). A similar reduction in foci was seen when PME-1was depleted with shRNA, suggesting that chemical inhibition of PME-1 isalso beneficial in endometrial cancer cell lines.

(c) PME-1 Inhibition Leads to Decreased Cell Migration

We next asked if PME-1 inhibition led to decreased cell migration bycompleting a trans-well migration assay. Cells were synchronized for 24hours in media containing 0% FBS. For this experiment, DMSO was againused as the vehicle control and Ishikawa cells were treated with DMSO(control) or 50 nM ABL-127 or 20 μM AMZ-30 for 24 hr and were allowed tomigrate through a collagen-coated well towards the bottom chamber,containing media with 30% FBS. As shown in FIG. 27, there is a dramaticdecrease in the percentage of cells that are capable of migrating whenPME-1 is inhibited with either covalent inhibitor. Taken together, thesedata suggest that PME-1 inhibition leads to decreased cancer phenotypes.

Example 21 Mice Study—Gene Silencing PME-1 Suppresses Tumor Formation

Because our data suggest that PME-1 promotes more aggressive EC, weasked if the over-expression of PME-1 in EC cells promoted the formationof tumors in a xenograft model. In this study, instead of RL95-2 cells,which require a larger number of cells to induce tumor formation, weused ECC-1 cells, which are aggressive endometrial adenocarcinoma cellswith high levels of endogenous PME-1 (FIG. 1) that have been usedpreviously for similar studies.

ECC-1 cells expressing either the empty vector (control) orover-expressing PME-1 (+PME-1) were subcutaneously injected into theflank of seven female immune-compromised mice per group and tumor sizewas measured weekly. Prior to injection, we performed Western blotanalysis to confirm the proper expression of PME-1 (FIG. 28A).

Mice injected with +PME-1 cells formed tumors with a significantlylarger tumor volume compared to control mice by 7 weeks post injection(FIG. 28B). Tumor bulks were visually larger when PME-1 was overexpressed (white bars represent 0.5 cm, FIG. 28C) and mean tumor weightwas increased in +PME-1 cells compared to control mice (FIG. 28D). Thesein vivo data correlate well with our in vitro data and furthersubstantiate our hypothesis that PME-1 promotes more aggressive cancergrowth and cell proliferation.

We next asked if depletion of PME-1 with shRNA led to decreased tumorgrowth over time. For this study, we subcutaneously injected 5×10⁶Ishikawa cells into the flank of female SCID mice and allowed the tumorsto grow until they reached a tumor volume of approximately 400 mm³. Oncetumors reached the appropriate size, we randomly divided the mice intotwo groups and injected them with adenovirus expressing either scrambledshRNA (Control-Ad, closed circles) or PPME1-shRNA (open circles). Tumorswere treated every three to four days with 5×10⁷ pfu (on days 0, 3, 7,10, and 13). As shown in FIG. 29, by day 13 there was a significantreduction in tumor volume when PME-1 was depleted with shRNA (opencircles) compared to control shRNA (closed circles). Taken together,these data suggest that the levels of PME-1 expression affect tumorgrowth and that depletion of PME-1 leads to decreased tumor growth.

Experimental Methods and Protocols

A) Maintenance of Cell Cultures

Cell lines were purchased from ATCC. RL95-2 cells were maintained inDulbecco Modified Eagle medium and Ham's F-12 supplement (DMEM:F12,Sigma Aldrich) with 10% fetal bovine serum (FBS, Sigma Aldrich), 5 μg/mlinsulin (Sigma Aldrich), and 100 μg/ml Primocin (InvivoGen). KLE cellswere maintained in Dulbecco Modified Eagle medium and Ham's F-12supplement (DMEM:F12) with 10% fetal bovine serum (FBS), and 100 μg/mlPrimocin. ECC-1 cells were maintained in RPMI-1640 medium (SigmaAldrich), supplemented with 10% FBS. Ishikawa cells . . . End1immortalized endocervical cells were maintained in keratinocyte-serumfree medium (KSFM, Life Technologies) with bovine pituitary extract,human epithelial growth factor (Life Technologies), and 10% FBSaccording to ATCC's recommendations.

B) Creation and Maintenance of Stable Cell Lines

N-terminal FLAG-tagged vector (p3XFLAG-CMV-10, cat. no. E7658;Signa-Aldrich, St. Louis, Mo.) were transfected into RL95-2 cells withLipofectamine 2000 (Life Technologies). The PME-1 gene was cloned intothe vector within the multi-cloning site with EcoR1 and BamH1restriction enzyme sites and inserted. Proper orientation of the genewas confirmed with sequencing, and empty vector was used as a control.

Lentivirus was produced in HEK293T cell lines with pLKO shRNA vectors(Thermo Scientific, cat. no. RHS4533; Waltham, Mass.) andpCDH-CuO-GFP-Puro vectors (System Biosciences, Inc., cat. no. QM513B-1;Mountain View, Calif.) with pPACKH1 packaging plasmid (SystemBiosciences, Inc., cat, no. LV050A-1). shRNA sequences are in the Table2. Lentivirus was collected and concentrated with Peg-IT concentratingsolution (System Biosciences, Inc.). 5×10⁶ ifu/ml lentivirus was addedto cell cultures with growth media and 8 μg/ml PolyBrene (SigmaAldrich). Media was changed to full growth media after 24 hrs and toselection media containing antibiotic after 48 hrs (5 μg/ml puromycin)(Invivogen) for 10 days.

C) RNA Extraction from Endometrial Cancer (EC) Patient Tissues

Matched pairs harvested from 30 patients with endometrial adenocarcinoma(see Table 1 for patient information) were used to determine the mRNAand protein levels of PME-1 in tumor versus normal adjacent tissue.Tissue samples were homogenized in 4 mL TRIzol Reagent (Invitrogen) onice and RNA and protein were extracted according to standard procedures.Briefly, samples were centrifuged at 10,000×g for 15 minutes at 4° C.and the supernatant was subjected to chloroform extraction. The aqueouslayer was subsequently used to isolate RNA by precipitation withisopropanol while the interphase/organic phase was saved at 4° C. forprotein extraction.

D) Protein Extraction from EC Patient Tissues

The protein was extracted by precipitation with acetone followed bycentrifugation at 12,000×g for 10 minutes at 4° C. The pellets werewashed with of 0.3M guanidine hydrochloride/95% ethanol/2.5% glycerol(v/v) solution and 2.5% glycerol/95% ethanol, air-dried, re-suspended in300 μl of 1% SDS and heated at 100° C. to solubilize protein. Proteinconcentrations were determined using Bio-Rad Protein Assay Dye ReagentConcentrate (Bio-Rad) on a SPECTRA Max plus spectrophotometer (MolecularDevices) at 595 nm wavelength.

E) qRT-PCR Analysis of Gene Expression in EC Patient Tissues

Gene expression analysis was conducted using the one-step SYBR green kit(Qiagen) as described in the manual. The following primers were used foramplification at 5 μM per reaction: PME1-Forward5′-AGGAAGGAAGTGAGTCTATAAG-3′ and PME-1-Reverse5′-CAGGTGTATGGATGGTCTT-3′. All data was normalized to 18S house-keepinggene. 18S primers were Forward 5′-AACCCGTTGAACCCCATT-3′ and Reverse5′-CCATCCAATCGGTAGTAGCG-3′. All data was normalized to RL95-2 cells tominimize plate-to-plate variance and fold-change is represented.

F) Taqman RT-PCR Analysis

Total RNA was extracted with Qiagen RNAEasy kit from RL95-2 cells. cDNAwas synthesized using 500 ng-1 μg of RNA and SuperScript VILO c-DNA mix(Life Technologies). Taqman probes and PCR-mix were purchased from LifeTechnologies. All samples were run in triplicates. Results arerepresentative of three independent experiments in which genes ofinterest were normalized to the house-keeping gene, 18S or GAPDH, wherefold-change was calculated using the ddCt method.

G) Western Analysis of EC Patient Tissues

15 μg of protein was used for western analysis. PME-1 antibody (SantaCruz) and COXIV (Cell Signaling Technologies) were used for detection ofproteins and the blots were developed using chemiluminescence (PierceECL Western Blotting Substrate—Thermo Scientific) on the GE ImageQuantLAS 4000. For analysis of Akt, ERK, and β-catenin signaling in tissueculture, western analyses were completed as follows. RL95-2 cells stablyinfected with lentivirus expressing constructs were selected withpuromycin (Invivogen). The cells were incubated with either 50 μM Aktinhibitor (LY 294002, Cell Signaling Technologies) or 40 μM ERKinhibitor (V0126, Cell Signaling Technologies) for 1-2 hours at 37° C.1.5× Laemelli buffer (0.5 M Tris pH6.8, 100% glycerol, 10% sodiumdodecyl sulfate, 100 mM EDTA) was used to prepare cell lysates. 20 μg ofprotein was used for western analysis. Primary antibodies used were:P-Akt (Ser473), P-Akt (Thr308), pan-Akt, P-ERK (Thr202/204), total ERK(Cell Signaling Technologies), PME-1 (Santa Cruz), phospho-β-catenin(Ser45, Cell Signaling Technologies), phospho-β-catenin (Ser33/37/Thr41,Cell Signaling Technologies), β-catenin (Abcam), DcR2 (Abcam), and GAPDH(Abcam). All experiments were repeated several times with similarresults.

H) Immunohistochemistry Analysis of EC Patient Tissues

Patient tissues representative of stages I, II and III endometrialadenocarcinoma and normal adjacent tissue were fixed for three hours onice in 4% PFA, followed by 30% sucrose in PBS and were then embedded inOCT (SAKURA), frozen on dry ice and sectioned to 10 μm slices using thecryostat (Microm HM 550). To stain, sections were incubated in methanol(Sigma Aldrich) with 0.3% hydrogen peroxide (Sigma Aldrich) to blockendogenous peroxidase, washed and blocked again in 10% FBS in TBST (TrisBuffered Saline with 0.1% Triton X-100). The sections were subsequentlyincubated with primary antibody over night, followed by detection usingVECTASTAIN Elite ABC biotyn-avidin-HRP kit (Vector Labs) and DAB. H&Estaining was performed according to standard protocol. Primaryantibodies include anti-PME-1 antibody (Antibodies-online).

I) Immunofluorescence Assays

For analysis of patient samples, tissues were blocked for 2 hr with 10%FBS in TBST for at RT, incubated with each primary antibody (together)overnight at 4° C., washed three times for 15 min in TBST, incubatedwith each secondary fluorescent antibody (together) for 1 hr at RT,washed again as above, stained with DAPI and mounted with cover-slipsusing vectashield mounting media (Vector Labs). Primary antibodies usedwere for PME-1 (Santa Cruz), E-cadherin (Millipore), and vimentin(Genscript). 1×10³RL95-2 cells were plated on a chamber slide and wereincubated overnight at 37° C. Slides were fixed with 4% paraformaldehyde(PFA) on ice for two hours. The cells were incubated with the primaryantibody to Histone 3 (pH3) phosphorylated at Ser10 (1:200, CellSignaling Technologies) overnight and developed with the secondaryanti-rabbit Alexa-Fluor 555 antibody (Cell Signaling Technologies). DAPIwas used as a nuclear counter-stain (Sigma Aldrich). Images were takenwith the Nikon Eclipse TE2000 at 20×.

J) Foci Formation Assays

1,000 cells were plated in a six well tissue culture dish in selectionmedia (to include 5 μg/ml puromycin) (InvivoGen). Cells were allowed toadhere and grow in a 5% CO₂, 37° C. incubator for 10 to 14 days. Mediawas replaced every fourth day. Cells were stained with 1% crystal violet(Sigma Aldrich) for five minutes and rinsed with PBS. Colonies werecounted and data is presented as the percentage of change compared tothe control. All Experiments were repeated at least three times.

K) TUNEL Assay

RL95-2 cells were plated in a chamber-slide and allowed to grow overnight at 37° C. Cells were transfected the next day with 100 nM of siRNA(Thermo Scientific) using Dharmafect 1 transfection reagent (siRNAsequences are summarized in Table S2). Transfections were repeated onday four and the cultures were terminated the day after. The assay wasperformed according to the manufacturer's instructions (Roche AppliedScience, counterstained with DAPI and photographed using Leica DM16000 Bmicroscope. Cells incubated with 1 U/μl of DNase I (Life Technologies)served as a positive control. The experiment was repeated three times.

L) Invasive Growth Assay in Matrigel

The 3D On Top Matrigel Assay was completed in 24 well dishes asdescribed previously {Lee, 2007 #1577}. Briefly, the bottoms of 24 welldishes were coated with 50 μl BD Matrigel Basement Matrix Phenol RedFree (BD Biosciences cat. #356237) and allowed to solidify at 37° C. 10⁵cells were suspended in 250 μl chilled medium containing either 10 ng/mlof TGF-b or vehicle and allowed to settle at 37° C. for 30 minutes.Chilled medium containing 10% of Matrigel was added by pipetting gentlydown the sidewall of the well and incubated at 37° C. for ten dayschanging top medium every second day. Colony counts were performed onthe tenth day, averages were taken from three fields of view using a 10×objective. Experiment was repeated three times.

M) Phosphatase Assay

Whole cell lysates were used with the DuoSet IC PP2A Phosphatase AssayKit (R&D Systems). Procedure was carried out as per kit instructionswith 500 μg protein and the absorbance was measured at 620 nm. The assaywas repeated twice with similar results.

N) In Vivo Tumor Formation

For the PME-1 over-expression in vivo study, 1×10⁶ endometrial carcinomacells (ECC-1) diluted in 100 μl 1×PBS expressing empty vector (Control)or over-expressing PME-1 (+PME-1) were injected subcutaneously into theflank of immune-compromised female mice (n=7 per group). Tumor formationwas measured weekly for seven weeks with a caliper and tumor volume wascalculated according to the formula V=½yx², where y=tumor length andx=tumor width. At 8 weeks post-injection, mice were euthanized andtumors were resected for analysis. Tumor masses were weighed and werehomogenized in TriZol reagent, as above, for protein extraction.

For the PME-1 depletion in vivo assay, 5×10⁶ Ishikawa cells and Matrigelwere subcutaneously injected into the flank of female SCID mice (n=14)and allowed the tumors to grow until they reached a tumor volume ofabout 400 mm³. Once tumors reached the appropriate size, mice wererandomly divided into two groups and were injected with adenovirusexpressing either scrambled shRNA (Control-Ad, closed circles) orPPME1-shRNA (open circles). Tumors were treated on days 0, 3, 7, 10, and13 with 5×10⁷ pfu. Tumors were measured as above and were resected forfurther analysis. Tumors were cut in half to complete western analysisand IHC.

All animal work was approved by and conducted according to theguidelines of the Genesis Biotechnology Group IACUC.

O) BrDU Incorporation Assay

The BrDU Incorporation assay was completed with RL95-2 cell lines inmedia supplemented with 2.5 μg/ml puromycin. Cells were plated at0.5*10^4 cells per well into a 96 well dish with 0% FBS and allowed tosettle for 24 hours. The assay was completed as per kit instructions(Roche, Cell Proliferation ELISA, BrdU colorimetric cat. #11647229001).Briefly, 10 uM BrdU was added to the cells, 24 hours later cells werefixed and denatured with FixDenat from the kit and allowed to incubatewith anti-BrdU antibody for up to two hours. Following the incubationand addition of the kit substrate, absorbances were read using FluoStarGalaxy.

P) Thermal Melt Assay

The assay was completed by first conducting a 10 min pre-incubation ofone μmole of recombinant PME-1des3 with 50 μM ABL127 (Sigma) or vehicle(0.05% DMSO) in the presence of SYBR Orange (1:1,000, v/v, Sigma) at 37°C. in 1×NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT,pH 7.9 at 25° C.), a thermal gradient was performed increasingtemperature by 2° C. per minute increments from 37° to 95° C. andfluorescence (492 nm-610 nm) was acquired on a thermal cycler Mx3005P(Stratagene).

Q) Trans-Well Migration Assay

The assay was completed with ECC-1 endometrial carcinoma cell linesfollowing kit instructions Cultrex 96 Well Collagen IV Cell InvasionAssay (cat #3458-096-K). Briefly, cells were serum starved for 24 hoursprior to experimentation, then counted and put into the top chamber of acollagen type IV coated Transwell membrane at a concentration of 1×10⁶cells/well in 0% FBS. The bottom chamber contained 30% FBS. Cells wereallowed to invade at 37° C. humid chamber incubator for 24 hours. Cellsthat invaded were analyzed using the kit provided Calcein-AM andfluorescence was read using FluoStar Galaxy.

R) Statistical Analysis

Statistical analysis was completed using GraphPad Prism software. Foranimal studies, significance was calculated using a two-way ANOVA. Forall other data, analysis was completed with the Mann-Whitney U test forsignificance (patient samples) or the student's standard t test;standard errors of the mean (SEM) were calculated for all samplebatches. The p values were represented as follows: *p<0.05, **p, 0.01,***p<0.001. ROC analysis plotting the sensitivity and specificity ofPME-1 mRNA levels in endometrial cancer patient samples (as shown inFIG. 3A) was calculated with 95% confidence interval to determine thevalidity of PME-1 as a biomarker for endometrial cancer using alikelihood ratio of 21. The determined p value (p<0.0001) and area underthe curve (AUC, 0.9601) suggests that PME-1 could be a valuablepredictor of endometrial cancer.

All publications and patents cited in this specification are hereinincorporated by reference in their entirety. Various modifications andvariations of the described composition, method, and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments andcertain working examples, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.

TABLE 1 Patient Information Patient FIGO TNM Tumor size ID Age GradeClass (cm) Metastases PPME1 06310 71 1 T3aN0M0 N/A ovary +14.42 06309 711 T1bN0M0 6.5 × 4 × 5 none +2.235 06308 57 1 T3aN0M0 N/A ovary +7.11106276 57 1 T1bN0M0 N/A ovary +7.945 06261 58 1 T3aN0M0 N/A none +24.9306273 69 1 T1aN0M0 N/A none +17.51 06272 79 1 T1bN0M0 N/A none +8.45606287 66 1 T1bN0M0 1 × 2 × 3.8 none −2.809 06313 58 1 T2N0M0 3.8 × 3.5 ×1 none +2.144 06314 70 1 T1bN0M0 5.5 × 6 × 2 none +4.724 06337 61 1T1bN0M0 3 × 3.5 none +2.329 06354 62 1 T1aN0M0 6 × 4.5 × 2 none +2.54906262 66 1 T1aN0M0 N/A none −2.219 06246 56 1-2 T1aN0M0 3 × 5 × 0.8 none+2.657 06240 59 2 T1bN0M0 3.5 × 2 × 1.5 none +5.242 06241 58 2 T1aN0M0N/A none +3.555 06245 82 2 T1bN0M0 N/A none +8.877 06247 64 2 T1aN0M05.5 × 1.5 × 1 none −5.856 06319 52 2 T1aN0M0 3.5 × 2.5 × 2 none +32.6706333 66 2 T1aN0M0 1.1 × 0.8 × 1 none +26.91 06336 56 2 T1bN0M0 7 × 6 ×3 none +2.402 06384 53 2 T2N0M0 6 × 5 × 2 none +9.987 06387 70 2 T1aN0M02.5 × 2.5 × 0.6 none +2.639 06269 61 2 T1aN0M0 0.4 none +30.48 06248 522 T1aN0M0 4 × 3.5 none −2.999 06268 65 3 T1bN0M0 4 × 2 none +12.47 0629441 3 T1aN0M0 N/A none +4.993 06338 58 3 T1aN0M0 5 × 4 x 1.5 none −33.1306457 63 3 T1aN0M0 8.5 × 7.5 × 3.5 none ND 06324 59 3 T1bN0M0 3 × 1.5 ×1.5 none +6.409 All patients were female, Caucasian, and were diagnosedwith Type I endometrial adenocarcinoma. All samples and patient reportswere analyzed by our in-house pathologist to determine stage and gradeof the tumors according to the International Federation of Gynecologyand Obstetrics (FIGO) guidelines. Tumor grades that are in bold font andunderlined could not be reassessed due to lack of remaining tumormaterial. The amount of PPME1 (the gene that codes for PME-1) normalizedto the 18S house-keeping gene is shown as the fold-increase for tumorcompared to NAT for each individual patient. ND = not determined

TABLE 2 RNAi sequences targeting PPME1, which codes for PME-1 proteinNon- Control shRNA Not disclosed targeting shRNA shPPME1 no. 1 SEQ IDNO: 1: GTA CAG bp 554-574 CTA TGG ATG CAC TTA shPPME1 no. 2 SEQ ID NO:2: GCA GCG bp 289-309 ATT ATT AGT AGA GTT shPPME1 no. 3 SEQ ID NO: 3:GGT GTT Bp 964-984 GAT AGA TTG GAT AAA Non- Control siRNA Not disclosedtargeting siRNA PPME1 siRNA no. 1 SEQ ID NO: 4: TGG CTG bp 959-977 GTGTTG ATA GAT T PPME1 siRNA no. 2 SEQ ID NO: 5: GTG GAT 3′-UTR AGC ATC ACAAGA A PPME1 siRNA no. 3 SEQ ID NO: 6: GTA AAT 3′-UTR ACG TCG CAC CAG ASequences are listed that target the PME-1 mRNA, which is 1,161 bp inlength (targeted base pairs (bp) are shown). Two exemplary siRNAs targetthe 3′UTR region of the mRNA. The full sequence for the short hairpinsused to target PPME1 are exemplary shPPME1 no.1: 5′- CCGG-GTA CAG CTATGG ATG CAC TTA-CTC GAG-TAA GTG CAT CCA TAG CTG TAC-TTTTT-3′ (SEQ ID NO:7), shPPME1 no.2: 5′-CCGG-GCA GCG ATT ATT AGT AGA GTT-CTC GAG-AAC TCTACT AAT AAT CGC TGC-TTTTT-3′ (SEQ ID NO: 8), and shPPME1 no. 3: 5′-CCGG- GGT GTT GAT AGA TTG GAT AAA — CTC GAG — TTT ATC CAA TCT ATC AACACC — TTT TTG (SEQ ID NO: 9).

What is claimed is:
 1. A method for determining whether a woman hasendometrial cancer, comprising the steps of: (a) obtaining anendometrial tissue from a woman suspected of having an increased risk ofendometrial cancer; (b) obtaining a normal endometrial tissue; (c)treating said endometrial tissue obtained in step (a) with a lysisbuffer to prepare a first lysate; (d) treating said endometrial tissuein obtain step (b) with a lysis buffer to prepare a second lysate; (e)performing a Western blot or an ELISA to quantify PME-1 proteinexpression level in said first lysate and PME-1 protein expression levelin said second lysate; (f) comparing said PME-1 protein expression levelin said first lysate with said PME-1 protein expression level in saidsecond lysate; and (g) diagnosing said woman as having endometrialcancer, if there is an increased PME-1 protein expression level in saidendometrial tissue obtained in step (a) relative to said normalendometrial tissue obtained in step (b), wherein said Western blot orELISA uses a goat, rabbit or mouse antibody.
 2. A method of detectingPME-1 in a patient, comprising the steps of: (a) obtaining anendometrial tissue from a woman suspected of having an increased risk ofendometrial cancer; (b) performing a Western blot or an ELISA to on theendometrial tissue to quantify PME-1 protein expression level in saidtissue.
 3. The method of claim 2 wherein said endometrial tissueobtained in step (a) is treated with a lysis buffer to prepare a lysate,and the Western Blot or ELISA is performed on the lysate.
 4. The methodof claim 3, wherein said lysis buffer is a modified RIPA solution. 5.The method of claim 2, wherein said step (b) is performed by a Westernblot.
 6. The method of claim 2, wherein said step (b) is performed by anELISA.
 7. The method of claim 5, wherein said Western blot is performedusing an anti-PME-1 monoclonal antibody or polyclonal antibody.
 8. Themethod of claim 6, wherein said ELISA is performed using an anti-PME-1monoclonal antibody or polyclonal antibody.
 9. The method of claim 2,wherein said endometrial tissue is grade II or grade III cancer tissue.10. The method of claim 2 wherein said Western blot or ELISA uses agoat, rabbit or mouse antibody.